Methods for breast cancer treatment

ABSTRACT

Disclosed herein are methods for treating subjects with breast cancer, comprising determining a therapeutic regimen for cancer by measuring the level (amount) of proteins of one or more biomarkers. Also disclosed are methods of predicting or assessing therapeutic outcome for subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage application under 35 U.S.C. § 371,and claims priority to and the benefit of the filing date ofInternational Application Number PCT/US2015/064437, filed Dec. 8, 2015,which claims priority to U.S. Provisional Patent Application No.62/089,120, filed Dec. 8, 2014, each of which is herein incorporated inits entirety.

FIELD OF THE INVENTION

Disclosed herein are methods for treating breast cancer comprisingpredicting or assessing the efficacy of a therapeutic regimen for breastcancer and administering an effective amount of a therapeutic agent totreat breast cancer in subjects. Specifically, disclosed are methods formeasuring biomarkers associated with positive treatment outcomes forsubjects with breast cancer.

BACKGROUND

Cancer is one of the leading causes of death in the world. Despiteimprovements in prevention, early detection, treatment and survival, theAmerican Cancer Society states that breast cancer is the second mostcommon newly diagnosed cancer and second leading cause of death amongwomen in the United States. Breast cancer is a cancer that forms intissues of the breast. The most common type of breast cancer is ductalcarcinoma, which begins in the lining of the milk ducts (thin tubes thatcarry milk from the lobules of the breast to the nipple). Another typeof breast cancer is lobular carcinoma, which begins in the lobules (milkglands) of the breast. Invasive breast cancer is breast cancer that hasspread from where it began in the breast ducts or lobules to surroundingnormal tissue. Breast cancer occurs in both men and women, although malebreast cancer is rare.

Targeted therapy is one treatment option available to patients. Targettherapy may involve the administration of drugs or macromolecules suchas antibodies that are selective for cancer cells and leave normal cellsrelatively unharmed. Conventional therapeutics for breast cancer arelimited in large part because it is not always possible to establish theefficacy of available drugs without trial and error. Further limitationsinclude one or more of the following: high production cost, drugresistance, complications resulting from chemotherapy; and/or safetyissues related to the use of the drug. Alternative approaches are neededfor improving targeted therapy for breast cancer.

Treatment options for breast cancer include surgery, radiation therapy,hormone therapy, chemotherapy and targeted therapy. Treatment regimensare often determined based on the stage of breast cancer, whether thetumor has hormone receptors, whether the tumor has too much HER2protein, general health, and other details such as the size of the tumorin relation to the size of the breast, or whether the subject has gonethrough menopause.

Many therapeutic agents and combinations of therapeutic agents are usedto treat breast cancer, and a challenging aspect of treatment isdetermining which therapeutic or therapeutic combination is most optimalfor treatment. Clinical studies continue to compare the most effectivetreatments against something that may be better. Common therapeuticsused for early breast cancer include the anthracyclines (such asdoxorubicin/Adriamycin® and epirubicin/Ellence®) and the taxanes (suchas paclitaxel/Taxol® and docetaxel/Taxotere®). These may be used incombination with certain other therapeutics, like fluorouracil (5-FU),cyclophosphamide (Cytoxan®), and carboplatin. For cancers that are HER2positive, the targeted therapeutic trastuzumab (Herceptin®) is oftengiven with one of the taxanes. Pertuzumab (Perjeta®) can also becombined with trastuzumab and docetaxel for HER2 positive cancers.

What is needed are methods and compositions for determining theselection of effective therapeutics for treating cancer such as breastcancer, and methods for treating subjects with cancer.

BRIEF SUMMARY

Disclosed herein are methods for treating breast cancer comprisingpredicting or assessing therapeutic efficacy of a therapeutic agent thattargets EGFR and HER2, including pan-HER inhibitors, the methodcomprising measuring protein levels of one or more biomarkers disclosedherein in cellular samples from subjects prior to treatment with thetherapeutic agent, comparing the measured protein levels of the one ormore biomarkers from the subjects to a baseline value for the proteinlevels of the respective one or more biomarkers, wherein an elevated ordecreased level of the proteins of the one or more biomarkers indicatesthat the subject is a responder to the therapeutic agent. Measurementsof proteins for methods disclosed herein may be made by proteindetection methods including but not limited to, RPPA,immunohistochemistry, ELISA, suspension bead array, mass spectrometry,dot blot, or western blot. In certain aspects, the proteins that aremeasured are phosphorylated. In certain aspects, the proteins that aremeasured are not phosphorylated. In certain aspects, methods comprisemeasurement of proteins of the one or more biomarkers, or combinationsthereof, disclosed herein, wherein the proteins may or may not bephosphorylated: wherein the one or more biomarkers comprise ALK.Y1586;ALK.Y1604; AMPKb1.S108; Caspase.7, cleaved D198; Cyclin.B1 total;Cyclin.D1.total; EGFR.Y1068, EGFR.Y1173, EGFR.Y992, eIF4G.S1108;IGFBP5.total; ERBB2.total; ERBB2.Y1248; ERBB2.Y877; ERBB3.Y1289;ERBB4.Y1284; FAK.Y576.Y577; JAK1.Y1022 and Y1023; JAK2.Y1007;MEK1.2.S217 and S211; MET.Y1234 and Y1235; p70S6K.T389; p70S6K.T412;PI3K.p85.Y458.p55. and Y199; AKT S473; AKT T308; EGFR total; EGFR Y1148;ERBB3 total; ERK1/2 T202/Y204; Heregulin total; mTOR S2448; mTOR total;PTEN S380; SHC Y317; PARP.total; PDGFRa.Y754; RET.Y905; RTK.ROR1.total;SHC.Y317; STAT5.Y694; VEGFR2.Y996; X4EBP1.S65; AMPKa1.S485; A.RAF.S299;IGF1R.Y1131. and IR.Y1146; MCSFR.Y732; A.RAF.S299; LC3B.total;TIE2.Y992; a JAK.STAT.pathway score (which is the sum of themeasurements for JAK1 Y1022/Y1023, JAK1 Y1007, STAT1 Y701 STAT2Y727,STAT3 Y705, STAT5 Y694), a mTOR pathway score (which is the sum of themeasurements for 4EBP1 S65; eIF4E S209; eIF4G S1108; eIF4G S1108; eIF4GS1108; mTOR S2448; p70S6K S371; p70S6K T389; p70S6K T412; S6RPS240/S244); a HER Family pathway score (which is the sum of themeasurements for EGFR Y1068; EGFR Y1173; EGFR Y992; ERBB2 Y1248; ERBB3Y1289; FAK Y576/Y577; SHC Y317; STAT5 Y694; RET Y905); and a RTK pathwayscore (which is the sum of the measurements for ALK Y1604; EGFR Y1068;EGFR Y1173; EGFR Y992; ERBB2 Y1248; ERBB3 Y1289; FAK Y576/Y577; SHCY317; STAT5 Y694; ERBB2 Y877; ERBB4 Y1284; MET Y1234-Y1235; ROS Y2274;RET Y905), or combinations thereof. As used herein with a biomarker,“total” means that the biomarker is the total amount of the particularprotein measured, which would include phosphorylated andnon-phosphorylated forms, if such forms exist, of the particularprotein. Measurements of proteins may be made by protein detectionmethods including but not limited to, RPPA, immunohistochemistry, ELISA,suspension bead array, mass spectrometry, dot blot, or western blot.

Disclosed herein are methods of treating breast cancer comprisingpredicting or assessing an increased likelihood of tumor shrinkage aftertreatment with a TKI (tyrosine kinase inhibitor) therapeutic agent thattargets EGFR and HER2, comprising a) measuring, in a pre-treatment tumorsample comprising cancer cells from a subject, protein levels of one ormore biomarkers disclosed herein, b) comparing the measured proteinlevels of the one or more biomarkers from the subjects to a baselinevalue for the respective protein levels of the one or more biomarkers,wherein an elevated or decreased level of the proteins of the respectiveone or more biomarkers indicates that the subject is a responder to thetherapeutic agent and has an increased likelihood of tumor shrinkageafter treatment with the TKI therapeutic agent, and administering aneffective amount of a therapeutic agent to treat the breast cancer. Oneor more biomarkers comprise, ALK.Y1586; ALK.Y1604; AMPKb1.S108;Caspase.7, cleaved D198; Cyclin.B1 total; Cyclin.D1.total; EGFR.Y1068,EGFR.Y1173, EGFR.Y992, eIF4G.S1108; IGFBP5.total; ERBB2.total;ERBB2.Y1248; ERBB2.Y877; ERBB3.Y1289; ERBB4.Y1284; FAK.Y576.Y577;JAK1.Y1022 and Y1023; JAK2.Y1007; MEK1.2.S217 and S211; MET.Y1234 andY1235; p70S6K.T389; p70S6K.T412; PI3K.p85.Y458.p55. and Y199; AKT S473;AKT T308; EGFR total; EGFR Y1148; ERBB3 total; ERK1/2 T202/Y204;Heregulin total; mTOR S2448; mTOR total; PTEN S380; SHC Y317;PARP.total; PDGFRa.Y754; RET.Y905; RTK.ROR1.total; SHC.Y317; STAT5.Y694;VEGFR2.Y996; X4EBP1.S65; AMPKa1.S485; A.RAF.S299; IGF1R.Y1131. andIR.Y1146; MCSFR.Y732; A.RAF.S299; LC3B.total; TIE2.Y992; aJAK.STAT.pathway score (which is the sum of the measurements for JAK1Y1022/Y1023, JAK1 Y1007, STAT1 Y701 STAT2Y727, STAT3 Y705, STAT5 Y694),a mTOR pathway score (which is the sum of the measurements for 4EBP1S65; eIF4E S209; eIF4G S1108; eIF4G S1108; eIF4G S1108; mTOR S2448;p70S6K S371; p70S6K T389; p70S6K T412; S6RP S240/S244); a HER Familypathway score (which is the sum of the measurements for EGFR Y1068; EGFRY1173; EGFR Y992; ERBB2 Y1248; ERBB3 Y1289; FAK Y576/Y577; SHC Y317;STAT5 Y694; RET Y905); and a RTK pathway score (which is the sum of themeasurements for ALK Y1604; EGFR Y1068; EGFR Y1173; EGFR Y992; ERBB2Y1248; ERBB3 Y1289; FAK Y576/Y577; SHC Y317; STAT5 Y694; ERBB2 Y877;ERBB4 Y1284; MET Y1234-Y1235; ROS Y2274; RET Y905), or combinationsthereof. Measurements of proteins may be made by protein detectionmethods including but not limited to, RPPA, immunohistochemistry, ELISA,suspension bead array, mass spectrometry, dot blot, or western blot Inan aspect, disclosed is a method for treating breast cancer comprisingpredicting or assessing an increased likelihood of tumor shrinkage aftertreatment with a TKI therapeutic agent that targets EGFR and HER2,comprising measuring, in a pre-treatment tumor sample comprising cancercells from a subject, protein levels of one or more biomarkers disclosedherein, comparing the measured protein levels of the one or morebiomarkers from the subjects to a baseline value for the respectiveprotein levels of the one or more biomarkers, wherein an elevated ordecreased level of the one or more biomarkers indicates that the subjectis a responder to the therapeutic agent and has an increased likelihoodof tumor shrinkage after treatment with the TKI therapeutic agent,wherein the one or more biomarkers comprise, AKT S473, AKT T308, EGFRtotal, EGFR Y1068, EGFR Y1148, EGFR Y1173, EGFR Y992, ERBB2 total, ERBB2Y1248, ERBB3 total, ERBB3 Y1289, ERK1/2 T202/Y204, Heregulin total, mTORS2448, mTOR total, PI3K p85 Y458/p55 Y199, PTEN S380, and SHC Y317, orcombinations thereof, and administering an effective amount of atherapeutic agent to treat the breast cancer. As used herein, a“pre-treatment tumor sample” means a sample obtained from a subjectbefore the subject is administered the particular therapeutic agent oragents under consideration, and does not mean that the subject has notpreviously been treated with chemotherapeutic agents or other therapies.A subject may have undergone one or more therapeutic regimens, and apre-treatment tumor sample is the sample obtained before theadministration of a therapeutic agent, such as a TKI that targets EGFRand/or HER2, or a pan-HER inhibitor, or a pan-EGFR inhibitor.

Disclosed are methods of treating cancer in a subject comprisingadministering a TKI that targets EGFR and HER2, wherein the subject hasbeen identified as having an elevated or decreased level (amount) ofproteins of one or more biomarkers, wherein the protein level measuredis compared to a baseline value for the proteins of the respective oneor more biomarkers, wherein the one or more biomarkers compriseALK.Y1586; ALK.Y1604; AMPKb1.S108; Caspase.7, cleaved D198; Cyclin.B1total; Cyclin.D1.total; EGFR.Y1068, EGFR.Y1173, EGFR.Y992, eIF4G.S1108;IGFBP5.total; ERBB2.total; ERBB2.Y1248; ERBB2.Y877; ERBB3.Y1289;ERBB4.Y1284; FAK.Y576.Y577; JAK1.Y1022 and Y1023; JAK2.Y1007;MEK1.2.S217 and S211; MET.Y1234 and Y1235; p70S6K.T389; p70S6K.T412;PI3K.p85.Y458.p55. and Y199; PARP.total; PDGFRa.Y754; RET.Y905;RTK.ROR1.total; SHC.Y317; STAT5.Y694; VEGFR2.Y996; X4EBP1.S65;AMPKa1.S485; A.RAF.S299; IGF1R.Y1131. and IR.Y1146; MCSFR.Y732;A.RAF.S299; LC3B.total; TIE2.Y992; a JAK.STAT.pathway score (which isthe sum of the measurements for JAK1 Y1022/Y1023, JAK1 Y1007, STAT1 Y701STAT2Y727, STAT3 Y705, STAT5 Y694), a mTOR pathway score (which is thesum of the measurements for 4EBP1 S65; eIF4E S209; eIF4G S1108; eIF4GS1108; eIF4G S1108; mTOR S2448; p70S6K S371; p70S6K T389; p70S6K T412;S6RP S240/S244); a HER Family pathway score (which is the sum of themeasurements for EGFR Y1068; EGFR Y1173; EGFR Y992; ERBB2 Y1248; ERBB3Y1289; FAK Y576/Y577; SHC Y317; STAT5 Y694; RET Y905); and a RTK pathwayscore (which is the sum of the measurements for ALK Y1604; EGFR Y1068;EGFR Y1173; EGFR Y992; ERBB2 Y1248; ERBB3 Y1289; FAK Y576/Y577; SHCY317; STAT5 Y694; ERBB2 Y877; ERBB4 Y1284; MET Y1234-Y1235; ROS Y2274;RET Y905); or combinations thereof, and administering an effectiveamount of a therapeutic agent to treat the breast cancer.

Disclosed are methods of treating cancer in a subject comprisingadministering a TKI that targets EGFR and HER2, wherein the subject hasbeen identified as having an elevated or decreased level (amount) ofproteins of one or more biomarkers, wherein the protein level measuredis compared to a baseline value for the proteins of the respective oneor more biomarkers, wherein the one or more biomarkers comprise AKTS473, AKT T308, EGFR total, EGFR Y1068, EGFR Y1148, EGFR Y1173, EGFRY992, ERBB2 total, ERBB2 Y1248, ERBB3 total, ERBB3 Y1289, ERK1/2T202/Y204, Heregulin total, mTOR S2448, mTOR total, PI3K p85 Y458/p55Y199, PTEN S380, and SHC Y317, or combinations thereof, in apre-treatment tumor sample comprising cancer cells obtained from thesubject, and administering an effective amount of a therapeutic agent totreat the breast cancer.

Disclosed are methods of treating cancer in a subject comprising, a)measuring in a pre-treatment tumor sample comprising cancer cells from asubject, protein levels of one or more biomarkers disclosed herein, b)comparing the measured protein levels of the one or more biomarkers fromthe subjects to a baseline value for the respective protein levels ofthe one or more biomarkers, wherein the one or more biomarkers compriseALK.Y1586; ALK.Y1604; AMPKb1. S108; Caspase.7, cleaved D198; Cyclin.B1total; Cyclin.D1.total; EGFR.Y1068, EGFR.Y1173, EGFR.Y992, eIF4G.S1108;IGFBP5.total; ERBB2.total; ERBB2.Y1248; ERBB2.Y877; ERBB3.Y1289;ERBB4.Y1284; FAK.Y576.Y577; JAK1.Y1022 and Y1023; JAK2.Y1007;MEK1.2.S217 and S211; MET.Y1234 and Y1235; p70S6K.T389; p70S6K.T412;PI3K.p85.Y458.p55. and Y199; PARP.total; PDGFRa.Y754; RET.Y905;RTK.ROR1.total; SHC.Y317; STAT5.Y694; VEGFR2.Y996; X4EBP1.S65;AMPKa1.S485; A.RAF.S299; IGF1R.Y1131. and IR.Y1146; MCSFR.Y732;A.RAF.S299; LC3B.total; TIE2.Y992; a JAK.STAT.pathway score (which isthe sum of the measurements for JAK.STAT and related proteins in theJAK.STATpathway), a mTOR pathway score (which is the sum of themeasurements for 4EBP1 S65; eIF4E S209; eIF4G S1108; eIF4G S1108; eIF4GS1108; mTOR S2448; p70S6K S371; p70S6K T389; p70S6K T412; S6RPS240/S244); a HER Family pathway score (which is the sum of themeasurements for EGFR Y1068; EGFR Y1173; EGFRY992; ERBB2 Y1248; ERBB3Y1289; FAK Y576/Y577; SHC Y317; STAT5 Y694; RET Y905); and a RTK pathwayscore (which is the sum of the measurements for ALK Y1604; EGFR Y1068;EGFR Y1173; EGFR Y992; ERBB2 Y1248; ERBB3 Y1289; FAK Y576/Y577; SHCY317; STAT5 Y694; ERBB2 Y877; ERBB4 Y1284; MET Y1234-Y1235; ROS Y2274;RET Y905); or combinations thereof, wherein an elevated or decreasedlevel of the one or more biomarkers indicates that the subject is aresponder to a therapeutic agent and has an increased likelihood oftumor shrinkage after treatment with the therapeutic agent; andadministering to the subject an effective amount of one or moretherapeutic agents, including but not limited to, a TKI, an agent thattargets EGFR and HER2, neratinib, affatinib, lapatinib and other knownpan-HER or pan-EGFR inhibitors. Methods disclosed herein may comprises aseries of active steps including first measuring the levels (amounts) ofone or more particular biomarkers to detect a change in one or morebiomarkers from the baseline measured biomarkers. A change in abiomarker may include, but is not limited to an elevation or reductionin a) amounts (levels) of particular individual proteins, b)combinations of particular proteins, c) phosphorylation of particularindividual proteins or combinations of proteins, and/or d) totalprotein; The change is recognized by comparing one or more measuredbiomarkers from the subject, such as those comprising the levels ofphosphorylation and/or total protein levels of the proteins selected, toexogenous reference standards and/or calibration standards (alsoreferred to herein as a baseline value, and by implication, eachbiomarker included in a method would have its particular baseline valuefor comparison). Other steps of methods disclosed herein may compriseinterpolating or extrapolating the data; generating a report thatdescribes the biomarkers, such as activation/phosphorylation proteinlevels or total protein levels, on a patient-by-patient basis; andproviding this report to another, such as to a physician, who then canuse this information for treating the subject with a therapeutic agent;and administering an effective amount of at least one therapeutic agentto the subject For example, a therapeutic agent may be a TKI thattargets EGFR and HER2, including, but not limited to, neratinib,affatinib, lapatinib and other known pan-HER or pan-EGFR inhibitors.

Disclosed are methods of treating cancer in a subject comprising, a)measuring, in a pre-treatment tumor sample comprising cancer cells froma subject, protein levels of one or more biomarkers disclosed herein, b)comparing the measured protein levels of the one or more biomarkers fromthe subjects to a baseline value for the respective protein levels ofthe one or more biomarkers, wherein the one or more biomarkers compriseAKT S473, AKT T308, EGFR total, EGFR Y1068, EGFR Y1148, EGFR Y1173, EGFRY992, ERBB2 total, ERBB2 Y1248, ERBB3 total, ERBB3 Y1289, ERK1/2T202/Y204, Heregulin total, mTOR S2448, mTOR total, PI3K p85 Y458/p55Y199, PTEN S380, and SHC Y317; or combinations thereof, wherein anelevated or decreased level of the one or more biomarkers indicates thatthe subject is a responder to a therapeutic agent and/or has anincreased likelihood of tumor shrinkage after treatment with thetherapeutic agent. The method further comprises treating the subjectwith the therapeutic agent. For example, the therapeutic agent may be aTKI that targets EGFR and HER2, including, but not limited to,neratinib, affatinib, lapatinib and other known pan-HER or pan-EGFRinhibitors.

Additional advantages of the disclosed methods will be set forth in partin the description which follows, and in part will be understood fromthe description, or may be learned by practice of the disclosed methods.The advantages of the disclosed methods will be realized and attained bymeans of the elements and combinations particularly pointed out in theappended claims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of thedisclosed method and compositions and together with the description,serve to explain the principles of the disclosed method andcompositions.

FIGS. 1A-G provide scatter plots of seven HER family signaling pathwaybiomarkers, comprising proteins and phosphoproteins, (FIG. 1 A—EGFRY1068, FIG. 1 B—EGFR Y1173, FIG. 1 C—EGFR Y992, FIG. 1 D—ERBB2 total,FIG. 1 E—ERBB2 Y1248, FIG. 1 F—ERBB3 Y1289, FIG. 1 G—SHC Y317) that werestatistically significantly elevated (p<0.05) in all subjects treatedwith neratinib (regardless of HER2 and HR status) who achieved pCR (i.e.pCR YES) and not in the matched control arm. The plots show the levelsof the measured proteins for the tumor cells that were obtained from thepre-treatment biopsy sample from HER2+ or HER2−, HR+ or HR− breastcancer patients.

FIGS. 2A-D are pCR distribution plots of patients who achieved responsein the neratinib (N) and control (C) arms. Area under the curve (AUC)values are shown, FIG. 2A is the distribution plot and AUC for theHER2+/HR− graduating population and FIG. 2B for the EGFR Y1773biomarker, showing superior AUC for pEGFR Y1773 compared to thepredicate HER2+/HR− group, which was determined by FISH/IHC testing.FIGS. 2C and D show superior AUC for additive or subtractive additionsof pEGFR Y1773 and HER2+/HR− subgroups.

FIG. 3 shows an example of a reverse phase protein microarray.

FIGS. 4A and 4B are scatter plots of all subjects, not divided by HR orHER2 phenotype, for biomarkers mTor pathway score (A) and HER pathwayscore (B).

FIGS. 5A-E are scatter plots of HER2+ phenotype subjects, for biomarkersmTor pathway score (A); HER pathway score (B); ALK Y1586 (C); p70S6KT412 (D); EGFR Y992 (E).

FIGS. 6A-D are scatterplots of HER2− phenotype subjects, for biomarkersALK Y1586 (A); EGFR Y1173 (B); RTK-ROR1 total (C); TIE2 Y992 (D).

FIGS. 7A-L show two-way scatter plots for HR−/HER2− subjects, controland treated subjects, for various biomarkers.

FIGS. 8A-D show two-way scatter plots for HR−/HER2− subjects, controland treated subjects, for various biomarkers.

FIGS. 9A-H show two-way scatter plots for HR+/HER2+ subjects, controland treated subjects, for various biomarkers.

FIG. 10A-J show two-way scatter plots for HR+/HER2+ subjects, controland treated subjects, for various biomarkers.

DETAILED DESCRIPTION

The disclosed methods and compositions may be understood more readily byreference to the following detailed description of particularembodiments and the Examples included therein and to the Figures andtheir previous and following descriptions.

It is understood that disclosed methods are not limited to theparticular methodology, protocols, and reagents described as these mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention which will belimited only by the appended claims.

Currently, treatment of Stage II/III breast cancer involves neoadjuvanttreatment (pre-surgical) with drug regimens that may include therapiestargeting specific molecular defects. Disclosed herein are novel methodsfor treatment of breast cancer comprising identifying biomarkers thatpredict response to therapeutic agents used in the pre-surgical stage oftreatment of cancer. Subjects providing samples (pre-treatment biopsies)for studies described herein were characterized based on measurement ofestrogen/hormone receptor (HR) and HER2 (c-erbB2), and were described byHER2−/HR−. HER2+/HR+. HER2+/HR−, HER2−/HR+ phenotypes. Response totreatment was determined by the amount of tumor shrinkage and a lack ofappreciable tumor evident at surgery. A response of tumor shrinkageand/or lack of tumor when examined (such as at surgery) is referred toherein as “complete pathological response” (pCR), and has recently beendeemed by the US FDA as an appropriate clinical endpoint for therapeuticapproval. Neratinib is a therapeutic agent with a mechanism of actionthat is as a tyrosine kinase inhibitor that binds to and inhibitsc-erbB2 and EGFR protein activity and the respective linked downstreampathways. Therapeutic agents, including affatinib, lapatinib and otherknown pan-HER or pan-EGFR inhibitors, have a similar mechanism of actionand are effective in the methods described herein. Methods describedherein may comprise obtaining measurements of the amounts proteins froma subject sample using a protein array technology developed by theinventors, and is referred to herein as “reverse phase proteinmicroarray” (RPPA). This microarray allows for quantitative measurementof the amount of proteins and/or phosphorylated proteins in the tumor orcancerous cells from pretreatment sample, for example from biopsyspecimens of women with Stage II/III breast cancer. Subjects who maybenefit from the methods disclosed herein are subjects who have a highrisk of recurrence and who may be treated with a tyrosine kinaseinhibitor (TKI), including but not limited to neratinib, in addition totreatment with other chemotherapeutic agents. In the studies disclosedherein neratinib showed statistically significant clinical response inthe HER2+/HR− subtype, and pCR was observed in every subtype regardlessof HER2/HR status.

Disclosed herein are methods for treating breast cancer comprisingtreating a subject identified as having breast cancer with at least onetherapeutic agent that targets EGFR and HER2, wherein the efficacy ofthe at least one therapeutic agent has been determined by predicting orassessing therapeutic efficacy of a therapeutic agent that targets EGFRand HER2, comprising a) measuring protein levels of one or morebiomarkers disclosed herein in cellular samples from subjects prior totreatment with the therapeutic agent, b) comparing the measured proteinlevels of the one or more biomarkers from the subjects to a baselinevalue for the protein levels of the respective one or more biomarkers,wherein an elevated or decreased level of the proteins of the one ormore biomarkers indicates that the subject is a responder to thetherapeutic agent, and further comprising, administering an effectiveamount of a therapeutic agent to treat the breast cancer.

A method for treating breast cancer comprises, administering to asubject identified as having breast cancer, at least one therapeuticagent that targets EGFR and HER2, wherein the efficacy of the at leastone therapeutic agent has been determined by a) measuring protein levelsof one or more biomarkers disclosed herein in cellular samples fromsubjects prior to treatment with the therapeutic agent, and b) comparingthe measured protein levels of the one or more biomarkers from thesubjects to a baseline value for the protein levels of the respectiveone or more biomarkers, wherein an elevated or decreased level of theproteins of the one or more biomarkers indicates that the subject is aresponder to the therapeutic agent, wherein the one or more biomarkerscomprise ALK.Y1586; ALK.Y1604; AMPKb1.S108; Caspase.7, cleaved D198;Cyclin.B1 total; Cyclin.D1.total; EGFR.Y1068, EGFR.Y1173, EGFR.Y992,eIF4G.S1108; IGFBP5.total; ERBB2.total; ERBB2.Y1248; ERBB2.Y877;ERBB3.Y1289; ERBB4.Y1284; FAK.Y576.Y577; JAK1.Y1022 and Y1023;JAK2.Y1007; MEK1.2.S217 and S211; MET.Y1234 and Y1235; p70S6K.T389;p70S6K.T412; PI3K.p85.Y458.p55. and Y199; AKT S473; AKT T308; EGFRtotal; EGFR Y1148; ERBB3 total; ERK1/2 T202/Y204; Heregulin total; mTORS2448; mTOR total; PTEN S380; SHC Y317; PARP.total; PDGFRa.Y754;RET.Y905; RTK.ROR1.total; SHC.Y317; STAT5.Y694; VEGFR2.Y996; X4EBP1.S65;AMPKa1.S485; A.RAF.S299; IGF1R.Y1131. and IR.Y1146; MCSFR.Y732;A.RAF.S299; LC3B.total; TIE2.Y992; a JAK.STAT.pathway score (which isthe sum of the measurements for JAK1 Y1022/Y1023, JAK1 Y1007, STAT1 Y701STAT2Y727, STAT3 Y705, STAT5 Y694), a mTOR pathway score (which is thesum of the measurements for 4EBP1 S65; eIF4E S209; eIF4G S1108; eIF4GS1108; eIF4G S1108; mTOR S2448; p70S6K S371; p70S6K T389; p70S6K T412;S6RP S240/S244); a HER Family pathway score (which is the sum of themeasurements for EGFR Y1068; EGFR Y1173; EGFR Y992; ERBB2 Y1248; ERBB3Y1289; FAK Y576/Y577; SHC Y317; STAT5 Y694; RET Y905); and a RTK pathwayscore (which is the sum of the measurements for ALK Y1604; EGFR Y1068;EGFR Y1173; EGFR Y992; ERBB2 Y1248; ERBB3 Y1289; FAK Y576/Y577; SHCY317; STAT5 Y694; ERBB2 Y877; ERBB4 Y1284; MET Y1234-Y1235; ROS Y2274;RET Y905), or combinations thereof. In certain aspects, the proteinsthat are measured are phosphorylated. In certain aspects, the proteinsthat are measured are not phosphorylated. In an aspect, methods fortreating breast cancer comprise administering to a subject identified ashaving breast cancer an effective amount of at least one therapeuticagent that targets EGFR and HER2, wherein determining the efficacy ofthe at least one therapeutic agent comprises predicting or assessingtherapeutic efficacy of a therapeutic agent that targets EGFR and HER2,comprise measurement of proteins of one or more biomarkers comprisingALK.Y1586; ALK.Y1604; AMPKb1.S108; Caspase.7, cleaved D198; Cyclin.B1total; Cyclin.D1.total; EGFR.Y1068, EGFR.Y1173, EGFR.Y992, eIF4G. S1108;IGFBP5.total; ERBB2.total; ERBB2.Y1248; ERBB2.Y877; ERBB3.Y1289;ERBB4.Y1284; FAK.Y576.Y577; JAK1.Y1022 and Y1023; JAK2.Y1007;MEK1.2.S217 and S211; MET.Y1234 and Y1235; p70S6K.T389; p70S6K.T412;PI3K.p85.Y458.p55. and Y199; AKT S473; AKT T308; EGFR total; EGFR Y1148;ERBB3 total; ERK1/2 T202/Y204; Heregulin total; mTOR S2448; mTOR total;PTEN S380; SHC Y317; PARP.total; PDGFRa.Y754; RET.Y905; RTK.ROR1.total;SHC.Y317; STAT5.Y694; VEGFR2.Y996; X4EBP1.S65; AMPKa1.S485; A.RAF.S299;IGF1R.Y1131. and IR.Y1146; MCSFR.Y732; A.RAF.S299; LC3B.total;TIE2.Y992; a JAK.STAT.pathway score (which is the sum of themeasurements for JAK1 Y1022/Y1023, JAK1 Y1007, STAT1 Y701 STAT2Y727,STAT3 Y705, STAT5 Y694), a mTOR pathway score (which is the sum of themeasurements for 4EBP1 S65; eIF4E S209; eIF4G S1108; eIF4G S1108; eIF4GS1108; mTOR S2448; p70S6K S371; p70S6K T389; p70S6K T412; S6RPS240/S244); a HER Family pathway score (which is the sum of themeasurements for EGFR Y1068; EGFR Y1173; EGFR Y992; ERBB2 Y1248; ERBB3Y1289; FAK Y576/Y577; SHC Y317; STAT5 Y694; RET Y905); and a RTK pathwayscore (which is the sum of the measurements for ALK Y1604; EGFR Y1068;EGFR Y1173; EGFR Y992; ERBB2 Y1248; ERBB3 Y1289; FAK Y576/Y577; SHCY317; STAT5 Y694; ERBB2 Y877; ERBB4 Y1284; MET Y1234-Y1235; ROS Y2274;RET Y905), or combinations thereof. As used herein with a biomarker,“total” means that the biomarker is the total amount of the particularprotein measured, which would include phosphorylated andnon-phosphorylated forms, if such forms exist, of the particularprotein. As used herein, “protein pathway activation module score” or“pathway score” were defined on the basis of known biochemical linkagesbetween the individual phosphoproteins, and for example, werequantitatively measured by RPPA, and could also be measured by proteindetection methods including but not limited to, RPPA,immunohistochemistry, ELISA, suspension bead array, mass spectrometry,dot blot, or western blot. Pathway activation module scores werecalculated by first scaling the relative intensity values within eachendpoint to the sample with the highest value, resulting in valuesranging from 1 to 0 that were designated as the “single endpoint score”.Second, final pathway activation module scores for each sample weregenerated by summing the single individual phosphoprotein score for eachendpoint component in a given module. These scores, referred to as a“protein pathway activation module score” or “pathway score” representthe entire activation status of each of the pathways in each patient.

In an aspect, methods for treating breast cancer comprisingadministering to a subject identified as having breast cancer aneffective amount of at least one therapeutic agent that targets EGFR andHER2, wherein the efficacy of the at least one therapeutic agent hasbeen determined by predicting or assessing therapeutic efficacy of atherapeutic agent that targets EGFR and HER2, comprise measurement ofproteins of one or more biomarkers comprising ALK.Y1586; Cyclin.B1.total; Cyclin.D1.PI3K.p85.Y458.p55.Y199; EGFR.Y1068; EGFR.Y1173;EGFR.Y992; eIF4G.S1108; ERBB2 total; ERBB2.Y1248; ERBB4.Y1284; RET.Y905;SHC.Y317; ERBB2.Y877; mTOR total; HER total; or RTK total; orcombinations thereof. Measurements may be made by RPPA for this methodand any other method disclosed herein. Measurements may be made usingprotein detection methods including but not limited to, RPPA,immunohistochemistry, ELISA, suspension bead array, mass spectrometry,dot blot, or western blot. In an aspect, methods for predicting orassessing or assessing the therapeutic efficacy of a therapeutic agentthat targets EGFR and HER2, comprise measurement of proteins of one ormore biomarkers comprising Cyclin.D1.total; IGFBP5.total; or PARP.total;or combinations thereof. In an aspect, methods for predicting orassessing therapeutic efficacy of a therapeutic agent that targets EGFRand HER2, comprise measurement of proteins of one or more biomarkerscomprising mTOR pathway activation score; HER family pathway activationscore; RTK pathway activation score; ALK.Y1586; EGFR.Y992; p70S6K.T412,or Cyclin.B1.total; or combinations thereof. In an aspect, methods forpredicting or assessing therapeutic efficacy of a therapeutic agent thattargets EGFR and HER2, comprise measurement of proteins of one or morebiomarkers comprising AKT S473; AKT T308; EGFR; EGFR Y1068; EGFR Y1148;EGFR Y1173; EGFR Y992; ERBB2; ERBB2 Y1248; ERBB3; ERBB3 Y1289; ERK1/2T202/Y204; Heregulin; mTOR; mTOR S2448; PI3K p85 Y458/p55 Y199; PTENS380; or SHC Y317; or combinations thereof. In an aspect, methods forpredicting or assessing therapeutic efficacy of a therapeutic agent thattargets EGFR and HER2, comprise measurement of proteins of one or morebiomarkers comprising EGFR Y1068; EGFR Y1173; EGFR Y992; ERBB2 total;ERBB2 Y1248; ERBB3 Y1289; SHC Y317; or combinations thereof. In anaspect, methods for predicting or assessing therapeutic efficacy of atherapeutic agent that targets EGFR and HER2, comprise measurement ofproteins of one or more biomarkers comprising IGFBP5.total. In anaspect, methods for predicting or assessing therapeutic efficacy of atherapeutic agent that targets EGFR and HER2, comprise measurement ofproteins of one or more biomarkers comprising EGFR.Y1173; ERBB2.Y1248;ERBB4.Y1284; PDGFRa.Y754; or RTK.ROR1 total; or combinations thereof. Inan aspect, methods for predicting or assessing therapeutic efficacy of atherapeutic agent that targets EGFR and HER2, comprise measurement ofproteins of one or more biomarkers comprising A.RAF.S299; LC3B.total; orTIE2.Y992; or combinations thereof. As used herein, “measurement ofproteins” includes measurement of the amount (i.e., mg or μg), thepresence or absence of a protein; whether a protein is phosphorylated ornot, or other alterations to a protein, and each of the measurements arecompared to the baseline or control status of the biomarker. Those ofskill in the art can establish a baseline or control reference point fora particular biomarker and such a baseline or control reference pointmay be derived from cancer cells or from normal (non-cancerous) cells.

Disclosed herein are methods of predicting or assessing an increasedlikelihood of tumor shrinkage after treatment with a TKI (tyrosinekinase inhibitor) therapeutic agent that targets EGFR and HER2,comprising a) measuring, in a pre-treatment tumor sample comprisingcancer cells from a subject, protein levels of one or more biomarkersdisclosed herein, b) comparing the measured protein levels of the one ormore biomarkers from the subjects to a baseline value for the respectiveprotein levels of the one or more biomarkers, wherein an elevated ordecreased level of the proteins of the respective one or more biomarkersindicates that the subject is a responder to the therapeutic agent andhas an increased likelihood of tumor shrinkage after treatment with theTKI therapeutic agent. One or more biomarkers comprise, ALK.Y1586;ALK.Y1604; AMPKb1.S108; Caspase.7, cleaved D198; Cyclin.B1 total;Cyclin.D1.total; EGFR.Y1068, EGFR.Y1173, EGFR.Y992, eIF4G.S1108;IGFBP5.total; ERBB2.total; ERBB2.Y1248; ERBB2.Y877; ERBB3.Y1289;ERBB4.Y1284; FAK.Y576.Y577; JAK1.Y1022 and Y1023; JAK2.Y1007;MEK1.2.S217 and S211; MET.Y1234 and Y1235; p70S6K.T389; p70S6K.T412;PI3K.p85.Y458.p55. and Y199; AKT S473; AKT T308; EGFR total; EGFR Y1148;ERBB3 total; ERK1/2 T202/Y204; Heregulin total; mTOR S2448; mTOR total;PTEN S380; SHC Y317; PARP.total; PDGFRa.Y754; RET.Y905; RTK.ROR1.total;SHC.Y317; STAT5.Y694; VEGFR2.Y996; X4EBP1.S65; AMPKa1.S485; A.RAF.S299;IGF1R.Y1131. and IR.Y1146; MCSFR.Y732; A.RAF.S299; LC3B.total;TIE2.Y992; a JAK.STAT.pathway score (which is the sum of themeasurements for JAK1 Y1022/Y1023, JAK1 Y1007, STAT1 Y701 STAT2Y727,STAT3 Y705, STAT5 Y694), a mTOR pathway score (which is the sum of themeasurements for 4EBP1 S65; eIF4E S209; eIF4G S1108; eIF4G S1108; eIF4GS1108; mTOR S2448; p70S6K S371; p70S6K T389; p70S6K T412; S6RPS240/S244); a HER Family pathway score (which is the sum of themeasurements for EGFR Y1068; EGFR Y1173; EGFR Y992; ERBB2 Y1248; ERBB3Y1289; FAK Y576/Y577; SHC Y317; STAT5 Y694; RET Y905); and a RTK pathwayscore (which is the sum of the measurements for ALK Y1604; EGFR Y1068;EGFR Y1173; EGFR Y992; ERBB2 Y1248; ERBB3 Y1289; FAK Y576/Y577; SHCY317; STAT5 Y694; ERBB2 Y877; ERBB4 Y1284; MET Y1234-Y1235; ROS Y2274;RET Y905), or combinations thereof.

In an aspect, a method for treating breast cancer comprises,administering to a subject identified as having breast cancer, at leastone therapeutic agent that targets EGFR and HER2, wherein the efficacyof the at least one therapeutic agent has been determined by predictingor assessing an increased likelihood of tumor shrinkage after treatmentwith a TKI therapeutic agent comprise measurement of proteins of one ormore biomarkers comprising ALK.Y1586; Cyclin.B1. total;Cyclin.D1.PI3K.p85.Y458.p55.Y199; EGFR.Y1068; EGFR.Y1173; EGFR.Y992;eIF4G.S1108; ERBB2 total; ERBB2.Y1248; ERBB4.Y1284; RET.Y905; SHC.Y317;ERBB2.Y877; mTOR total; HER total; or RTK total; or combinationsthereof. In an aspect, a method for treating breast cancer comprises,administering to a subject identified as having breast cancer, at leastone therapeutic agent that targets EGFR and HER2, wherein the efficacyof the at least one therapeutic agent has been determined by predictingor assessing an increased likelihood of tumor shrinkage after treatmentwith a TKI therapeutic agent comprise measurement of proteins of one ormore biomarkers comprising Cyclin.D1.total; IGFBP5.total; or PARP.total;or combinations thereof. In an aspect, a method for treating breastcancer comprises, administering to a subject identified as having breastcancer, at least one therapeutic agent that targets EGFR and HER2,wherein the efficacy of the at least one therapeutic agent has beendetermined by predicting or assessing an increased likelihood of tumorshrinkage after treatment with a TKI therapeutic agent comprisemeasurement of proteins of one or more biomarkers comprising mTORpathway activation.score; HER Family pathway activation score; RTKpathway activation score; ALK.Y1586; EGFR.Y992; p70S6K.T412, orCyclin.B1.total; or combinations thereof. In an aspect, a method fortreating breast cancer comprises, administering to a subject identifiedas having breast cancer, at least one therapeutic agent that targetsEGFR and HER2, wherein the efficacy of the at least one therapeuticagent has been determined by predicting or assessing an increasedlikelihood of tumor shrinkage after treatment with a TKI therapeuticagent comprise measurement of proteins of one or more biomarkerscomprising AKT S473; AKT T308; EGFR; EGFR Y1068; EGFR Y1148; EGFR Y1173;EGFR Y992; ERBB2; ERBB2 Y1248; ERBB3; ERBB3 Y1289; ERK1/2 T202/Y204;Heregulin; mTOR; mTOR S2448; PI3K p85 Y458/p55 Y199; PTEN S380; or SHCY317; or combinations thereof. In an aspect, a method for treatingbreast cancer comprises, administering to a subject identified as havingbreast cancer, at least one therapeutic agent that targets EGFR andHER2, wherein the efficacy of the at least one therapeutic agent hasbeen determined by predicting or assessing an increased likelihood oftumor shrinkage after treatment with a TKI therapeutic agent comprisemeasurement of proteins of one or more biomarkers comprising EGFR Y1068;EGFR Y1173; EGFR Y992; ERBB2 total; ERBB2 Y1248; ERBB3 Y1289; SHC Y317;or combinations thereof. In an aspect, a method for treating breastcancer comprises, administering to a subject identified as having breastcancer, at least one therapeutic agent that targets EGFR and HER2,wherein the efficacy of the at least one therapeutic agent has beendetermined by predicting or assessing an increased likelihood of tumorshrinkage after treatment with a TKI therapeutic agent comprisemeasurement of proteins of one or more biomarkers comprisingIGFBP5.total. In an aspect a method for treating breast cancercomprises, administering to a subject identified as having breastcancer, at least one therapeutic agent that targets EGFR and HER2,wherein the efficacy of the at least one therapeutic agent has beendetermined by predicting or assessing an increased likelihood of tumorshrinkage after treatment with a TKI therapeutic agent comprisemeasurement of proteins of one or more biomarkers comprising EGFR.Y1173;ERBB2.Y1248; ERBB4.Y1284; PDGFRa.Y754; or RTK.ROR1.total; orcombinations thereof. In an aspect, a method for treating breast cancercomprises, administering to a subject identified as having breastcancer, at least one therapeutic agent that targets EGFR and HER2,wherein the efficacy of the at least one therapeutic agent has beendetermined by predicting or assessing an increased likelihood of tumorshrinkage after treatment with a TKI therapeutic agent comprisemeasurement of proteins of one or more biomarkers comprising A.RAF.S299;LC3B.total; or TIE2.Y992; or combinations thereof.

Disclosed are methods of treating cancer in a subject comprisingadministering an effective amount of a TKI that targets EGFR and HER2,wherein the subject has been identified as having an elevated ordecreased level (amount) of proteins of one or more biomarkers, whereinthe protein level measured is compared to a baseline value for theproteins of the respective one or more biomarkers, wherein the one ormore biomarkers comprise ALK.Y1586; ALK.Y1604; AMPKb1.S108; Caspase.7,cleaved D198; Cyclin.B1 total; Cyclin.D1.total; EGFR.Y1068, EGFR.Y1173,EGFR.Y992, eIF4G. S1108; IGFBP5.total; ERBB2.total; ERBB2.Y1248;ERBB2.Y877; ERBB3.Y1289; ERBB4.Y1284; FAK.Y576.Y577; JAK1.Y1022 andY1023; JAK2.Y1007; MEK1.2.S217 and S211; MET.Y1234 and Y1235;p70S6K.T389; p70S6K.T412; PI3K.p85.Y458.p55. and Y199; AKT S473; AKTT308; EGFR total; EGFR Y1148; ERBB3 total; ERK1/2 T202/Y204; Heregulintotal; mTOR S2448; mTOR total; PTEN S380; SHC Y317; PARP.total;PDGFRa.Y754; RET.Y905; RTK.ROR1.total; SHC.Y317; STAT5.Y694;VEGFR2.Y996; X4EBP1.S65; AMPKa1.S485; A.RAF.S299; IGF1R.Y1131. andIR.Y1146; MCSFR.Y732; A.RAF.S299; LC3B.total; TIE2.Y992; aJAK.STAT.pathway score (which is the sum of the measurements for JAK1Y1022/Y1023, JAK1 Y1007, STAT1 Y701 STAT2Y727, STAT3 Y705, STAT5 Y694),a mTOR pathway score (which is the sum of the measurements for 4EBP1S65; eIF4E S209; eIF4G S1108; eIF4G S1108; eIF4G S1108; mTOR S2448;p70S6K S371; p70S6K T389; p70S6K T412; S6RP S240/S244); a HER Familypathway score (which is the sum of the measurements for EGFR Y1068; EGFRY1173; EGFR Y992; ERBB2 Y1248; ERBB3 Y1289; FAK Y576/Y577; SHC Y317;STAT5 Y694; RET Y905); and a RTK pathway score (which is the sum of themeasurements for ALK Y1604; EGFR Y1068; EGFR Y1173; EGFR Y992; ERBB2Y1248; ERBB3 Y1289; FAK Y576/Y577; SHC Y317; STAT5 Y694; ERBB2 Y877;ERBB4 Y1284; MET Y1234-Y1235; ROS Y2274; RET Y905), or combinationsthereof. For example, the therapeutic agent may be a TKI that targetsEGFR and HER2, including, but not limited to, neratinib, affatinib,lapatinib and other known pan-HER or pan-EGFR inhibitors. Measurementsmay be made by protein detection methods including but not limited to,RPPA, immunohistochemistry, ELISA, suspension bead array, massspectrometry, dot blot, or western blot.

In an aspect, a method of treating cancer in a subject comprisingadministering an effective amount of a TKI that targets EGFR and HER2,wherein the subject has been identified as having an elevated ordecreased level (amount) of proteins of one or more biomarkers in apre-treatment tumor sample comprising cancer cells obtained from thesubject, comprises measurement of proteins of one or more biomarkerscomprising ALK.Y1586; Cyclin.B1. total;Cyclin.D1.PI3K.p85.Y458.p55.Y199; EGFR.Y1068; EGFR.Y1173; EGFR.Y992;eIF4G.S1108; ERBB2 total; ERBB2.Y1248; ERBB4.Y1284; RET.Y905; SHC.Y317;ERBB2.Y877; mTOR total; HER total; or RTK total; or combinationsthereof. For example, the therapeutic agent may be a TKI that targetsEGFR and HER2, including, but not limited to, neratinib, affatinib,lapatinib and other known pan-HER or pan-EGFR inhibitors. Measurementsmay be made by protein detection methods including but not limited to,RPPA, immunohistochemistry, ELISA, suspension bead array, massspectrometry, dot blot, or western blot. In an aspect, a method oftreating cancer in a subject comprising administering an effectiveamount of one or more of TKI that targets EGFR and HER2, wherein thesubject has been identified as having an elevated or decreased level(amount) of proteins of one or more biomarkers in a pre-treatment tumorsample comprising cancer cells obtained from the subject, comprisesmeasurement of proteins of one or more biomarkers comprisingCyclin.D1.total; IGFBP5.total; or PARP.total; or combinations thereof.For example, the therapeutic agent may be a TKI that targets EGFR andHER2, including, but not limited to, neratinib, affatinib, lapatinib andother known pan-HER or pan-EGFR inhibitors. Measurements may be made byprotein detection methods including but not limited to, RPPA,immunohistochemistry, ELISA, suspension bead array, mass spectrometry,dot blot, or western blot. In an aspect, a method of treating cancer ina subject comprising administering an effective amount of one or more ofa TKI that targets EGFR and HER2, wherein the subject has beenidentified as having an elevated or decreased level (amount) of proteinsof one or more biomarkers in a pre-treatment tumor sample comprisingcancer cells obtained from the subject, comprises measurement ofproteins of one or more biomarkers comprising mTOR pathwayactivation.score; HER Family pathway activation score; RTK pathwayactivation score; ALK.Y1586; EGFR.Y992; p70S6K.T412, or Cyclin.B1.total;or combinations thereof. For example, the therapeutic agent may be a TKIthat targets EGFR and HER2, including, but not limited to, neratinib,affatinib, lapatinib and other known pan-HER or pan-EGFR inhibitors.Measurements may be made by protein detection methods including but notlimited to, RPPA, immunohistochemistry, ELISA, suspension bead array,mass spectrometry, dot blot, or western blot. In an aspect, a method oftreating cancer in a subject comprising administering an effectiveamount of one or more of a TKI that targets EGFR and HER2, wherein thesubject has been identified as having an elevated or decreased level(amount) of proteins of one or more biomarkers in a pre-treatment tumorsample comprising cancer cells obtained from the subject, comprisesmeasurement of proteins of one or more biomarkers comprising AKT S473;AKT T308; EGFR; EGFR Y1068; EGFR Y1148; EGFR Y1173; EGFR Y992; ERBB2;ERBB2 Y1248; ERBB3; ERBB3 Y1289; ERK1/2 T202/Y204; Heregulin; mTOR; mTORS2448; PI3K p85 Y458/p55 Y199; PTEN S380; or SHC Y317; or combinationsthereof. For example, the therapeutic agent may be a TKI that targetsEGFR and HER2, including, but not limited to, neratinib, affatinib,lapatinib and other known pan-HER or pan-EGFR inhibitors. Measurementsmay be made by protein detection methods including but not limited to,RPPA, immunohistochemistry, ELISA, suspension bead array, massspectrometry, dot blot, or western blot. In an aspect, a method oftreating cancer in a subject comprising administering an effectiveamount of one or more of a TKI that targets EGFR and HER2, wherein thesubject has been identified as having an elevated or decreased level(amount) of proteins of one or more biomarkers in a pre-treatment tumorsample comprising cancer cells obtained from the subject, comprisesmeasurement of proteins of one or more biomarkers comprising EGFR Y1068;EGFR Y1173; EGFR Y992; ERBB2 total; ERBB2 Y1248; ERBB3 Y1289; SHC Y317;or combinations thereof. For example, the therapeutic agent may be a TKIthat targets EGFR and HER2, including, but not limited to, neratinib,affatinib, lapatinib and other known pan-HER or pan-EGFR inhibitors.Measurements may be made by protein detection methods including but notlimited to, RPPA, immunohistochemistry, ELISA, suspension bead array,mass spectrometry, dot blot, or western blot. In an aspect, a method oftreating cancer in a subject comprising administering an effectiveamount of one or more of a TKI that targets EGFR and HER2, wherein thesubject has been identified as having an elevated or decreased level(amount) of proteins of one or more biomarkers in a pre-treatment tumorsample comprising cancer cells obtained from the subject, comprisesmeasurement of proteins of one or more biomarkers comprisingIGFBP5.total. For example, the therapeutic agent may be a TKI thattargets EGFR and HER2, including, but not limited to, neratinib,affatinib, lapatinib and other known pan-HER or pan-EGFR inhibitors.Measurements may be made by protein detection methods including but notlimited to, RPPA, immunohistochemistry, ELISA, suspension bead array,mass spectrometry, dot blot, or western blot. In an aspect, a method oftreating cancer in a subject comprising administering an effectiveamount of one or more of a TKI that targets EGFR and HER2, wherein thesubject has been identified as having an elevated or decreased level(amount) of proteins of one or more biomarkers in a pre-treatment tumorsample comprising cancer cells obtained from the subject, comprisesmeasurement of proteins of one or more biomarkers comprising EGFR.Y1173;ERBB2.Y1248; ERBB4.Y1284; PDGFRa.Y754; or RTK.ROR1.total; orcombinations thereof. For example, the therapeutic agent may be a TKIthat targets EGFR and HER2, including, but not limited to, neratinib,affatinib, lapatinib and other known pan-HER or pan-EGFR inhibitors.Measurements may be made by protein detection methods including but notlimited to, RPPA, immunohistochemistry, ELISA, suspension bead array,mass spectrometry, dot blot, or western blot. In an aspect, a method oftreating cancer in a subject comprising administering an effectiveamount of one or more of a TKI that targets EGFR and HER2, wherein thesubject has been identified as having an elevated or decreased level(amount) of proteins of one or more biomarkers in a pre-treatment tumorsample comprising cancer cells obtained from the subject, comprisesmeasurement of proteins of one or more biomarkers comprising A.RAF.S299;LC3B.total; or TIE2.Y992; or combinations thereof. For example, thetherapeutic agent may be a TKI that targets EGFR and HER2, including,but not limited to, neratinib, affatinib, lapatinib and other knownpan-HER or pan-EGFR inhibitors. Measurements may be made by proteindetection methods including but not limited to, RPPA,immunohistochemistry, ELISA, suspension bead array, mass spectrometry,dot blot, or western blot.

Disclosed are methods of treating cancer in a subject comprising, a)measuring in a pre-treatment tumor sample comprising cancer cells from asubject, protein levels of one or more biomarkers disclosed herein, b)comparing the measured protein levels of the one or more biomarkers fromthe subjects to a baseline value for the respective protein levels ofthe one or more biomarkers, wherein the one or more biomarkers compriseALK.Y1586; ALK.Y1604; AMPKb1.S108; Caspase.7, cleaved D198; Cyclin.B1total; Cyclin.D1.total; EGFR.Y1068, EGFR.Y1173, EGFR.Y992, eIF4G.S1108;IGFBP5.total; ERBB2.total; ERBB2.Y1248; ERBB2.Y877; ERBB3.Y1289;ERBB4.Y1284; FAK.Y576.Y577; JAK1.Y1022 and Y1023; JAK2.Y1007;MEK1.2.S217 and S211; MET.Y1234 and Y1235; p70S6K.T389; p70S6K.T412;PI3K.p85.Y458.p55. and Y199; AKT S473; AKT T308; EGFR total; EGFR Y1148;ERBB3 total; ERK1/2 T202/Y204; Heregulin total; mTOR S2448; mTOR total;PTEN S380; SHC Y317; PARP.total; PDGFRa.Y754; RET.Y905; RTK.ROR1.total;SHC.Y317; STAT5.Y694; VEGFR2.Y996; X4EBP1.S65; AMPKa1.S485; A.RAF.S299;IGF1R.Y1131. and IR.Y1146; MCSFR.Y732; A.RAF.S299; LC3B.total;TIE2.Y992; a JAK.STAT.pathway score (which is the sum of themeasurements for JAK1 Y1022/Y1023, JAK1 Y1007, STAT1 Y701 STAT2Y727,STAT3 Y705, STAT5 Y694), a mTOR pathway score (which is the sum of themeasurements for 4EBP1 S65; eIF4E S209; eIF4G S1108; eIF4G S1108; eIF4GS1108; mTOR S2448; p70S6K S371; p70S6K T389; p70S6K T412; S6RPS240/S244); a HER Family pathway score (which is the sum of themeasurements for EGFR Y1068; EGFR Y1173; EGFR Y992; ERBB2 Y1248; ERBB3Y1289; FAK Y576/Y577; SHC Y317; STAT5 Y694; RET Y905); and a RTK pathwayscore (which is the sum of the measurements for ALK Y1604; EGFR Y1068;EGFR Y1173; EGFR Y992; ERBB2 Y1248; ERBB3 Y1289; FAK Y576/Y577; SHCY317; STAT5 Y694; ERBB2 Y877; ERBB4 Y1284; MET Y1234-Y1235; ROS Y2274;RET Y905), or combinations thereof, wherein an elevated or decreasedlevel of the one or more biomarkers indicates that the subject is aresponder to a therapeutic agent and has an increased likelihood oftumor shrinkage after treatment with the therapeutic agent. In anaspect, a method may further comprise administering to the subject aneffective amount of the therapeutic agent. For example, the therapeuticagent may be a TKI that targets EGFR and HER2, including, but notlimited to, neratinib, affatinib, lapatinib and other known pan-HER orpan-EGFR inhibitors. Measurements may be made by protein detectionmethods including but not limited to, RPPA, immunohistochemistry, ELISA,suspension bead array, mass spectrometry, dot blot, or western blot.

In an aspect, a method of treating cancer in a subject comprising, a)measuring in a pre-treatment tumor sample comprising cancer cells from asubject, protein levels of one or more biomarkers disclosed herein, b)comparing the measured protein levels of the one or more biomarkers fromthe subjects to a baseline value for the respective protein levels ofthe one or more biomarkers, wherein the one or more biomarkers compriseALK.Y1586; Cyclin.B1. total; Cyclin.D1.PI3K.p85.Y458.p55.Y199;EGFR.Y1068; EGFR.Y1173; EGFR.Y992; eIF4G.S1108; ERBB2 total;ERBB2.Y1248; ERBB4.Y1284; RET.Y905; SHC.Y317; ERBB2.Y877; mTOR total;HER total; or RTK total; or combinations thereof, and further comprisingadministering an effective amount of one of more TKIs that target EGFRand HER2, including, but not limited to, neratinib, affatinib, lapatiniband other known pan-HER or pan-EGFR inhibitors. Measurements may be madeby protein detection methods including but not limited to, RPPA,immunohistochemistry, ELISA, suspension bead array, mass spectrometry,dot blot, or western blot. In an aspect, a method of treating cancer ina subject comprising, a) measuring in a pre-treatment tumor samplecomprising cancer cells from a subject, protein levels of one or morebiomarkers disclosed herein, b) comparing the measured protein levels ofthe one or more biomarkers from the subjects to a baseline value for therespective protein levels of the one or more biomarkers, wherein the oneor more biomarkers comprise Cyclin.D1.total; IGFBP5.total; orPARP.total; or combinations thereof; and further comprisingadministering an effective amount of one of more TKIs that target EGFRand HER2, including, but not limited to, neratinib, affatinib, lapatiniband other known pan-HER or pan-EGFR inhibitors. Measurements may be madeby R protein detection methods including but not limited to, RPPA,immunohistochemistry, ELISA, suspension bead array, mass spectrometry,dot blot, or western blot. In an aspect, a method of treating cancer ina subject comprising, a) measuring in a pre-treatment tumor samplecomprising cancer cells from a subject, protein levels of one or morebiomarkers disclosed herein, b) comparing the measured protein levels ofthe one or more biomarkers from the subjects to a baseline value for therespective protein levels of the one or more biomarkers, wherein the oneor more biomarkers comprise mTOR pathway activation.score; HER Familypathway activation score; RTK pathway activation score; ALK.Y1586;EGFR.Y992; p70S6K.T412, or Cyclin.B1.total; or combinations thereof, andfurther comprising administering an effective amount of one of more TKIsthat target EGFR and HER2, including, but not limited to, neratinib,affatinib, lapatinib and other known pan-HER or pan-EGFR inhibitors.Measurements may be made by protein detection methods including but notlimited to, RPPA, immunohistochemistry, ELISA, suspension bead array,mass spectrometry, dot blot, or western blot. In an aspect, a method oftreating cancer in a subject comprising, a) measuring in a pre-treatmenttumor sample comprising cancer cells from a subject, protein levels ofone or more biomarkers disclosed herein, b) comparing the measuredprotein levels of the one or more biomarkers from the subjects to abaseline value for the respective protein levels of the one or morebiomarkers, wherein the one or more biomarkers comprise AKT S473; AKTT308; EGFR; EGFR Y1068; EGFR Y1148; EGFR Y1173; EGFR Y992; ERBB2; ERBB2Y1248; ERBB3; ERBB3 Y1289; ERK1/2 T202/Y204; Heregulin; mTOR; mTORS2448; PI3K p85 Y458/p55 Y199; PTEN S380; or SHC Y317; or combinationsthereof, and further comprising administering an effective amount of oneof more TKIs that target EGFR and HER2, including, but not limited to,neratinib, affatinib, lapatinib and other known pan-HER or pan-EGFRinhibitors. Measurements may be made by protein detection methodsincluding but not limited to, RPPA, immunohistochemistry, ELISA,suspension bead array, mass spectrometry, dot blot, or western blot. Inan aspect, a method of treating cancer in a subject comprising, a)measuring in a pre-treatment tumor sample comprising cancer cells from asubject, protein levels of one or more biomarkers disclosed herein, b)comparing the measured protein levels of the one or more biomarkers fromthe subjects to a baseline value for the respective protein levels ofthe one or more biomarkers, wherein the one or more biomarkers compriseEGFR Y1068; EGFR Y1173; EGFR Y992; ERBB2 total; ERBB2 Y1248; ERBB3Y1289; SHC Y317; or combinations thereof, and further comprisingadministering an effective amount of one of more TKIs that target EGFRand HER2, including, but not limited to, neratinib, affatinib, lapatiniband other known pan-HER or pan-EGFR inhibitors. Measurements may be madeby protein detection methods including but not limited to, RPPA,immunohistochemistry, ELISA, suspension bead array, mass spectrometry,dot blot, or western blot. In an aspect, a method of treating cancer ina subject comprising, a) measuring in a pre-treatment tumor samplecomprising cancer cells from a subject, protein levels of one or morebiomarkers disclosed herein, b) comparing the measured protein levels ofthe one or more biomarkers from the subjects to a baseline value for therespective protein levels of the one or more biomarkers, wherein the oneor more biomarkers comprise IGFBP5 total, and further comprisingadministering an effective amount of one of more TKIs that target EGFRand HER2, including, but not limited to, neratinib, affatinib, lapatiniband other known pan-HER or pan-EGFR inhibitors. Measurements may be madeby protein detection methods including but not limited to, RPPA,immunohistochemistry, ELISA, suspension bead array, mass spectrometry,dot blot, or western blot. In an aspect, a method of treating cancer ina subject comprising, a) measuring in a pre-treatment tumor samplecomprising cancer cells from a subject, protein levels of one or morebiomarkers disclosed herein, b) comparing the measured protein levels ofthe one or more biomarkers from the subjects to a baseline value for therespective protein levels of the one or more biomarkers, wherein the oneor more biomarkers comprise EGFR.Y1173; ERBB2.Y1248; ERBB4.Y1284;PDGFRa.Y754; or RTK.ROR1.total; or combinations thereof, and furthercomprising administering an effective amount of one of more TKIs thattarget EGFR and HER2, including, but not limited to, neratinib,affatinib, lapatinib and other known pan-HER or pan-EGFR inhibitors.Measurements may be made by protein detection methods including but notlimited to, RPPA, immunohistochemistry, ELISA, suspension bead array,mass spectrometry, dot blot, or western blot. In an aspect, a method oftreating cancer in a subject comprising, a) measuring in a pre-treatmenttumor sample comprising cancer cells from a subject, protein levels ofone or more biomarkers disclosed herein, b) comparing the measuredprotein levels of the one or more biomarkers from the subjects to abaseline value for the respective protein levels of the one or morebiomarkers, wherein the one or more biomarkers comprise A.RAF.S299;LC3B.total; or TIE2.Y992; or combinations thereof, and furthercomprising administering an effective amount of one of more TKIs thattarget EGFR and HER2, including, but not limited to, neratinib,affatinib, lapatinib and other known pan-HER or pan-EGFR inhibitors.Measurements may be made by protein detection methods including but notlimited to, RPPA, immunohistochemistry, ELISA, suspension bead array,mass spectrometry, dot blot, or western blot. Such methods comprisewherein an elevated or decreased level of the one or more biomarkersindicates that the subject is a responder to a therapeutic agent and hasan increased likelihood of tumor shrinkage after treatment with thetherapeutic agent. The method may further comprise administering to thesubject an effective amount of the therapeutic agent. For example, thetherapeutic agent may be a TKI that targets EGFR and HER2, including,but not limited to, neratinib, affatinib, lapatinib and other knownpan-HER or pan-EGFR inhibitors.

While data are specifically presented for predicting or assessing tumorshrinkage in breast cancer patients treated with neratinib, thephosphoproteins identified as predictive markers are ubiquitously foundin every tumor type including lung, colon, gastric, rectal, ovarian,pancreatic, prostate, brain, melanomas, sarcomas, etc as well asleukemias, myelomoas and lymphomas. Given the recent findings that drugefficacy transcends tumor type or cancer type, but is based onunderpinning drug target expression (eg. HER2 therapy success forHER2+/mutated lung, gastric and prostate cancers), it would be rationalto expect the markers described herein would be useful for predicting orassessing response in these other tumor types. Moreover, it would bereasonable to expect that these specific predictive phosphoproteinswould be predictive for tumor shrinkage and clinical response fortherapeutics that have similar mechanisms of action such as Gilotrif™(affatinib) and Tykerb™ (lapatinib).

Data presented herein demonstrate that the measurement of specificphosphorylation sites such as Y1173 and Y1068 on EGFR were predictive ofneratinib response, given the coincident phosphorylation events drivenby kinase-substrate interactions and domain based (e.g. SH2-SH3)protein-protein interactions, one could reasonably expect that otherprotein phosphorylation sites on the proteins described herein would bepredictive for treatment response/tumor shrinkage.

For example, with some phenotypes or no particular phenotype, particularbiomarker(s) were found to be useful in treating subjects and inpredicting or assessing outcome (pCR). The followingprotein/phosphoproteins levels for biomarkers were statisticallysignificantly (p<0.05) higher in ALL patients (without pre-selection byHER2/HR testing) who received neratinib and achieved pCR versus thosewho did not, and were not different between response groups in thecontrol arm of the trial: ALK.Y1586; Cyclin.B1. total;Cyclin.D1.PI3K.p85.Y458.p55.Y199; EGFR.Y1068; EGFR.Y1173; EGFR.Y992;eIF4G.S1108; ERBB2 total; ERBB2.Y1248; ERBB4.Y1284; RET.Y905; SHC.Y317;ERBB2.Y877; mTOR total; HER total; or RTK total; or combinationsthereof.

The following proteins/phosphoproteins for biomarkers were statisticallysignificantly (p<0.05) lower in ALL patients (without pre-selection byHER2/HR testing) who received neratinib and achieved pCR versus thosewho did not and were not different between response groups in thecontrol arm of the trial: Cyclin.D1.total; IGFBP5.total; or PARP.total;or combinations thereof.

The following proteins/phosphoprotein levels for biomarkers werestatistically significantly (p<0.05) higher in all HER2+ patients whoreceived neratinib without pre-selection by HR testing and achieved pCRvs those who didn't, and were not different between response groups inpatients who did not receive the drug: mTOR pathway activation.score;HER Family pathway activation score; RTK pathway activation score;ALK.Y1586; EGFR.Y992; p70S6K.T412, or Cyclin.B1.total; or combinationsthereof. The following proteins/phosphoprotein levels were statisticallysignificantly (p<0.05) higher in all HER2+/HR+ patients who receivedneratinib and achieved pCR versus those who did not, and not differentbetween response groups in patients who did not receive the drug:ALK.Y1586; ALK.Y1604; AMPKb1.S108; Caspase.7, cleaved D198; Cyclin.B1total; Cyclin.D1.total; EGFR.Y1068, EGFR.Y1173, EGFR.Y992, eIF4G. S1108;IGFBP5.total; ERBB2.total; ERBB2.Y1248; ERBB2.Y877; ERBB3.Y1289;ERBB4.Y1284; FAK.Y576.Y577; JAK1.Y1022 and Y1023; JAK2.Y1007;MEK1.2.S217 and S211; MET.Y1234 and Y1235; p70S6K.T389; p70S6K.T412;PI3K.p85.Y458.p55. and Y199; AKT S473; AKT T308; EGFR total; EGFR Y1148;ERBB3 total; ERK1/2 T202/Y204; Heregulin total; mTOR S2448; mTOR total;PTEN S380; SHC Y317; PARP.total; PDGFRa.Y754; RET.Y905; RTK.ROR1.total;SHC.Y317; STAT5.Y694; VEGFR2.Y996; X4EBP1.S65; AMPKa1.S485; A.RAF.S299;IGF1R.Y1131. and IR.Y1146; MCSFR.Y732; A.RAF.S299; LC3B.total;TIE2.Y992; a JAK.STAT.pathway score (which is the sum of themeasurements for JAK1 Y1022/Y1023, JAK1 Y1007, STAT1 Y701 STAT2Y727,STAT3 Y705, STAT5 Y694), a mTOR pathway score (which is the sum of themeasurements for 4EBP1 S65; eIF4E S209; eIF4G S1108; eIF4G S1108; eIF4GS1108; mTOR S2448; p70S6K S371; p70S6K T389; p70S6K T412; S6RPS240/S244); a HER Family pathway score (which is the sum of themeasurements for EGFR Y1068; EGFR Y1173; EGFR Y992; ERBB2 Y1248; ERBB3Y1289; FAK Y576/Y577; SHC Y317; STAT5 Y694; RET Y905); and a RTK pathwayscore (which is the sum of the measurements for ALK Y1604; EGFR Y1068;EGFR Y1173; EGFR Y992; ERBB2 Y1248; ERBB3 Y1289; FAK Y576/Y577; SHCY317; STAT5 Y694; ERBB2 Y877; ERBB4 Y1284; MET Y1234-Y1235; ROS Y2274;RET Y905), or combinations thereof. The following protein for thebiomarker was lower in HER2+/HR+ patients who received neratinib andachieved pCR and not between response groups in patients who did notreceive neratinib: IGFBP5.total. The following proteins/phosphoproteinsfor biomarkers were statistically significantly (p<0.05)activated/over-expressed in all HER2−/HR− patients who receivedneratinib and achieved pCR and not between response groups in patientswho did not receive the drug: EGFR.Y1173; ERBB2.Y1248; ERBB4.Y1284;PDGFRa.Y754; or RTK.ROR1.total; or combinations thereof. The followingproteins/phosphoproteins for biomarkers were statistically significantly(p<0.05) lower in HER2−/HR− patients who received neratinib and achievedpCR and were not different between response groups in patients who didnot receive the drug: A.RAF.S299; LC3B.total; or TIE2.Y992; orcombinations thereof.

In methods described herein, administration or delivery of thetherapeutic agents to cells can be via one or more modes ofadministration or formulations. For example, the therapeutic agent canbe formulated as a pharmaceutical composition. Additionally, dosingregimens and dose amounts can be determined by methods known to those ofskill in the art. Pharmaceutical compositions can be administered in anumber of ways depending on whether local or systemic treatment isdesired, and on the area to be treated.

Preparations of parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Formulations for optical administration can include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Compositions fororal administration include powders or granules, suspensions orsolutions in water or non-aqueous media, capsules, sachets, or tablets.Thickeners, flavorings, diluents, emulsifiers, dispersing aids, orbinders may be desirable. Some of the compositions can be administeredas a pharmaceutically acceptable acid- or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines.

Methods disclosed herein comprise detection of proteins of biomarkersdisclosed herein. The detection methods used may include reverse phasemicroarray analysis, or any other methods known for protein detectionknown to those skilled in the art.

While the reverse phase protein microarray (RPPA) technology was used asa discovery platform that uncovered predictive biomarkers disclosedherein and methods to better select and treat patients for neratinibtreatment, there are a number of proteomic technologies that could beused to measure the specific markers for clinical implementation. Suchtechnologies include, but are not limited to, suspension bead arrays,immunohistochemistry, mass cytometry, mass spectrometry, ELISA, westernblotting, etc. Detection methods could be colorimetric, florescent,chemiluminescent, electrochemical or any routinely used means ofmeasuring proteins from a tissue sample. Use of detection methods arewithin the knowledge of those of skill in the art.

An aspect of the invention is a kit for predicting or assessing asubject's response to a therapeutic agent and/or the subject's prognosisor prediction of tumor shrinkage, comprising one or more agents formeasuring the level of protein of one or more biomarkers disclosedherein. The agents can be, e.g., antibodies specific for phosphorylatedor non-phosphorylated forms of the proteins. The kit may include agentssuitable for a label or label-free method known in the art to measureprotein levels of one or more biomarkers disclosed herein, such asagents for using mass spectrometry or electrophoretic mobility.Biomarkers may comprise one or more of ALK.Y1586; ALK.Y1604;AMPKb1.S108; Caspase.7, cleaved D198; Cyclin.B1 total; Cyclin.D1.total;EGFR.Y1068, EGFR.Y1173, EGFR.Y992, eIF4G.S1108; IGFBP5.total;ERBB2.total; ERBB2.Y1248; ERBB2.Y877; ERBB3.Y1289; ERBB4.Y1284;FAK.Y576.Y577; JAK1.Y1022 and Y1023; JAK2.Y1007; MEK1.2.S217 and S211;MET.Y1234 and Y1235; p70S6K.T389; p70S6K.T412; PI3K.p85.Y458.p55. andY199; AKT S473; AKT T308; EGFR total; EGFR Y1148; ERBB3 total; ERK1/2T202/Y204; Heregulin total; mTOR S2448; mTOR total; PTEN S380; SHC Y317;PARP.total; PDGFRa.Y754; RET.Y905; RTK.ROR1.total; SHC.Y317; STAT5.Y694;VEGFR2.Y996; X4EBP1.S65; AMPKa1.S485; A.RAF.S299; IGF1R.Y1131. andIR.Y1146; MCSFR.Y732; A.RAF.S299; LC3B.total; TIE2.Y992; aJAK.STAT.pathway score (which is the sum of the measurements for JAK1Y1022/Y1023, JAK1 Y1007, STAT1 Y701 STAT2Y727, STAT3 Y705, STAT5 Y694),a mTOR pathway score (which is the sum of the measurements for 4EBP1S65; eIF4E S209; eIF4G S1108; eIF4G S1108; eIF4G S1108; mTOR S2448;p70S6K S371; p70S6K T389; p70S6K T412; S6RP S240/S244); a HER Familypathway score (which is the sum of the measurements for EGFR Y1068; EGFRY1173; EGFR Y992; ERBB2 Y1248; ERBB3 Y1289; FAK Y576/Y577; SHC Y317;STAT5 Y694; RET Y905); and a RTK pathway score (which is the sum of themeasurements for ALK Y1604; EGFR Y1068; EGFR Y1173; EGFR Y992; ERBB2Y1248; ERBB3 Y1289; FAK Y576/Y577; SHC Y317; STAT5 Y694; ERBB2 Y877;ERBB4 Y1284; MET Y1234-Y1235; ROS Y2274; RET Y905), or combinationsthereof.

An aspect of the invention is a pharmaceutical composition, or a kit fortreating a subject in need thereof, comprising a TKI that targets EGFRand HER2. Pharmaceutical compositions comprise a pharmaceuticallyacceptable carrier. The pharmaceutical agent or kit may further comprisea chemotherapeutic agent that can be administered in conjunction withthe that targets EGFR and HER2. Such kits disclosed may be combined soas to provide the pharmaceutical composition and agents for detectingthe desired biomarkers.

Definitions

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “atherapeutic” includes a plurality of such therapeutics, reference to“the breast” is a reference to one or more breast cancer associatedcomplications known to those skilled in the art, and so forth.

The term “dose” refers to the quantity or amount of a composition taken,administered, or recommended to be taken or administered at or over aparticular time. The time can be per administration or per day. Forexample, a dose of a therapeutic can be, but is not limited to, aspecific amount of a composition, such as an immunosuppressant,administered 2× a day. A dose can also be a specific amount of acomposition administered every day for 2 weeks.

As used herein, “cancer” is meant to mean any of many diseasescharacterized by the presence of cancerous tissue in a subject. As usedherein, “cancerous tissue” is meant to mean a tissue that comprisesmalignant neoplastic cells, exhibits an abnormal growth of cells and/orhyperproliferative cells. Cancerous tissue can be a primary malignanttumor, arising in a tissue or organ of origin, or it can be a metastaticmalignant tumor, growing in a body tissue which was not the source ofthe original tumor. Thus, malignant neoplastic cells can invade anddestroy nearby tissue and spread to other parts of the body(metastasize). As used herein, the term “neoplastic” means an abnormalgrowth of a cell or tissue (e.g., a tumor or non-solid hyperproliferative cellular activity) which may be benign or malignant(cancerous). As used herein, “abnormal growth of cells” and/or“hyperproliferative cells” are meant to refer to cell growth independentof normal regulatory mechanisms (e.g., loss of contact inhibition),including the abnormal growth of benign and malignant cells or otherneoplastic diseases. As used herein, the term “tumor” includes neoplasmsthat are identifiable through clinical screening or diagnosticprocedures including, but not limited to, palpation, biopsy, cellproliferation index, endoscopy, ultrasonography, computed tomography(CT), magnetic resonance imaging (MRI), positron emission tomography(PET), radiography, radionuclide evaluation, CT- or MRI-guidedaspiration cytology, and imaging-guided needle biopsy, among others.Such diagnostic techniques are well known to those skilled in the artand are described in Holland, et al., Cancer Medicine, 4th Ed., Vol.One, Williams & Wilkins, Baltimore, Md. (1997).

A “breast cancer” is a cancer that occurs in the breast of a subject.Breast cancer is a tumor (that may be malignant) that starts in thecells of the breast. A malignant tumor is a group of cancer cells thatcan grow into (invade) surrounding tissues or spread (metastasize) todistant areas of the body. The disease occurs almost entirely in women,but men can get it, too. Examples of breast cancer include, but are notlimited to, invasive ductal carcinoma, invasive lobular carcinoma,ductal carcinoma in situ, and lobular carcinoma in situ.

The term “efficacy” refers to the ability to produce a desired orintended result. For example, the efficacy of a therapeutic is theability of the therapeutic to produce the intended result, such as treata particular disease. Efficacy can be determined by evaluatinglaboratory tests, imaging results, signs and symptoms known to be usefulin evaluating the status of breast cancer. In some situations, efficacymay be determined as observation of tumor shrinkage such as with MRI, ora definitive histological/pathological determination such as “completepathological response”, or pCR or residual cancer burden levels (RCB)wherein no or little tumor is present at the time of surgery duringneoadjuvant treatment.

The term “therapeutic agent” refers to a composition that treats adisease. For example, the therapeutic agents disclosed herein arecompositions that treat breast cancer.

The phrase “therapeutic effect” refers to the treatment, reduction ofsymptoms amelioration, prevention of disease, inhibition of diseaseprogression, or reduced severity or incidence of disease as a result ofthe administration of a therapeutic.

The term “treating” refers to partially or completely alleviating,ameliorating, relieving, delaying onset of, inhibiting progression of,reducing severity of, and/or reducing incidence of one or more symptomsor features of a particular disease, disorder, and/or condition.Treatment may be administered to a subject who does not exhibit signs ofa disease, disorder, and/or condition and/or to a subject who exhibitsonly early signs of a disease, disorder, and/or condition for thepurpose of decreasing the risk of developing pathology associated withthe disease, disorder, and/or condition.

Neratinib is an orally available low molecular weight potentirreversible tyrosine kinase inhibitor, or TKI, that blocks signaltransduction through the epidermal growth factor receptors, or EGFRs,HER1, HER2 and HER4.

“Optional” or “optionally” means that the subsequently described event,circumstance, or material may or may not occur or be present, and thatthe description includes instances where the event, circumstance, ormaterial occurs or is present and instances where it does not occur oris not present.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, also specifically contemplated and considered disclosed isthe range

from the one particular value and/or to the other particular valueunless the context specifically indicates otherwise. Similarly, whenvalues are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms another,specifically contemplated embodiment that should be considered disclosedunless the context specifically indicates otherwise. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint unless the context specifically indicates otherwise. Finally,it should be understood that all of the individual values and sub-rangesof values contained within an explicitly disclosed range are alsospecifically contemplated and should be considered disclosed unless thecontext specifically indicates otherwise. The foregoing appliesregardless of whether in particular cases some or all of theseembodiments are explicitly disclosed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed method and compositions belong. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present method andcompositions, the particularly useful methods, devices, and materialsare as described.

Publications cited herein and the material for which they are cited arehereby specifically incorporated by reference. Nothing herein is to beconstrued as an admission that the present invention is not entitled toantedate such disclosure by virtue of prior invention. No admission ismade that any reference constitutes prior art. The discussion ofreferences states what their authors assert, and applicants reserve theright to challenge the accuracy and pertinency of the cited documents.It will be clearly understood that, although a number of publicationsare referred to herein, such reference does not constitute an admissionthat any of these documents forms part of the common general knowledgein the art.

Throughout the description and claims of this specification, the word“comprise” and variations of the word, such as “comprising” and“comprises,” means “including but not limited to,” and is not intendedto exclude, for example, other additives, components, integers or steps.In particular, in methods stated as comprising one or more steps oroperations it is specifically contemplated that each step comprises whatis listed (unless that step includes a limiting term such as “consistingof”), meaning that each step is not intended to exclude, for example,other additives, components, integers or steps that are not listed inthe step.

It is to be understood that the disclosed method and compositions arenot limited to specific synthetic methods, specific analyticaltechniques, or to particular reagents unless otherwise specified, and,as such, may vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only andis not intended to be limiting.

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed method and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutation of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. For example, if neratinib is disclosed and discussed and anumber of modifications that can be made to the drug are discussed, eachand every combination and permutation of the drug and the modificationsthat are possible are specifically contemplated unless specificallyindicated to the contrary. Thus, if a class of molecules A, B, and C aredisclosed as well as a class of molecules D, E, and F and an example ofa combination molecule, A-D is disclosed, then even if each is notindividually recited, each is individually and collectivelycontemplated. Thus, in this example, each of the combinations A-E, A-F,B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated andshould be considered disclosed from disclosure of A, B, and C; D, E, andF; and the example combination A-D. Likewise, any subset or combinationof these is also specifically contemplated and disclosed. Thus, forexample, the sub-group of A-E, B-F, and C-E are specificallycontemplated and should be considered disclosed from disclosure of A, B,and C; D, E, and F; and the example combination A-D. This conceptapplies to all aspects of this application including, but not limitedto, steps in methods of making and using the disclosed compositions.Thus, if there are a variety of additional steps that can be performedit is understood that each of these additional steps can be performedwith any specific embodiment or combination of embodiments of thedisclosed methods, and that each such combination is specificallycontemplated and should be considered disclosed.

A “subject,” as used herein, includes any animal, mammal, including ahuman, that has a cancer. Suitable subjects (patients) includelaboratory animals (such as mouse, rat, rabbit, or guinea pig), farmanimals, and domestic animals or pets (such as a cat or dog). Non-humanprimates and, human patients, are included.

A “baseline value,” as used herein, refers to the level or amount of thesame protein(s) or phosphoprotein(s) that are the biomarker, but thelevel or amount of protein(s) or phosphoprotein(s) are measured in cellsfrom tumors obtained from control subjects such as women who did notachieve pCR, or an optimized “cut-point” value determined by statisticalanalysis such as receiver operating curve (ROC) of a population of dataderived from tumors of patients who did or did not achieve clinicalsuccess (such as pCR or tumor shrinkage), or other relevant controls. Anincrease in the amount of a phosphoprotein can reflect either anincrease in the number of suitable amino acid residues of the protein(e.g., serines, threonines or tyrosines) that are phosphorylated, or anincreased frequency of phosphorylations at a particular amino acidresidue. For example, a baseline value may include reference standards,where a predetermined threshold value (or range of values) determineswhether the amount of measured protein, or the phosphorylation state ofthe protein, is above the “normal” value. The terms threshold level,control and baseline value are used interchangeably herein. For eachprotein whose level (amount) is determined, the value can be normalizedto the total protein in the cell; or to the amount of a constitutivelyexpressed protein (from a housekeeping gene), such as actin. Increasedamounts or decreased amounts of total protein, particular proteins orphosphorylated proteins can be determined routinely. For example,reference standards can be used, where a predetermined threshold value(or range of values) determines whether the amount of measured proteinis above the “baseline” value. Such a threshold value is sometimesreferred to herein as a baseline value.

REFERENCES

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EXAMPLES Example 1

Evaluation of HER Family Protein Signaling Network as a PredictiveBiomarker for pCR for Breast Cancer Patients Treated with Neratinib inthe I-SPY 2 TRIAL

Adaptive design trials are constructed on the basis of assigningpatients to certain signature groups such as signature biomarker groups,the goal being to determine predictive probabilities of success in PhaseIII trials for various targeted therapeutics. In essence, the designtrials provide data that facilitates the determination of which patientbiomarker signatures respond to various regimens. A regimen would“graduate” from trial if probability of statistical significance inPhase III reaches 85% for any signature, provided that a minimum of 60patients have been assigned to the regimen. Neratinib was one of 7agents tested in the experimental arms of the ISPY-2 TRIAL to date.

This example tested the hypothesis that the response to the pan-ERBBinhibitor, neratinib, was predicted by pre-treatment HER2-EGFRsignaling. In the I-SPY 2 TRIAL, neratinib graduated in the HR−/HER2+signature. All patients received at least standard chemotherapy (whichwas comprised of 12 weekly cycles of paclitaxel (a taxane) therapyfollowed by 4 cycles of doxorubicin/cyclophosphamide (AC) therapy). ForHER2+ patients, neratinib was administered in place of trastuzumab, andall HER2+ control arm patients received chemotherapy plus trastuzumab.18 HER family signaling proteins were evaluated as biomarkers ofneratinib response using reverse phase protein microarray (RPMA) datafrom pre-treatment laser capture microdissection (LCM) purified tumorepithelium. Reverse phase protein microarray (RPPA) was used toquantitatively measure the level of proteins and phosphorylated proteinsin the pretreatment biopsy specimens of women with Stage breast cancerwho have a high risk of recurrence and who were treated withneratinib+chemotherapy vs women who were treated with chemotherapyalone.

One hundred sixty-eight (168) patients (neratinib: 106, concurrentcontrols: 62) had RPMA and pathological complete response (pCR) data.Eighteen (18) protein/phosphoproteins biomarkers relating to HER familysignaling were evaluated: AKT S473, AKT T308, EGFR, EGFR Y1068, EGFRY1148, EGFR Y1173, EGFR Y992, ERBB2, ERBB2 Y1248, ERBB3 total, ERBB3Y1289, ERK1/2 T202/Y204, Heregulin, mTOR, mTOR S2448, PI3K p85 Y458/p55Y199, PTEN S380, and SHC Y317. This study assessed association betweenbiomarker and response in the neratinib and control arms alone(likelihood ratio test), and relative performance between arms(biomarker×treatment interaction) using a logistic model. Analysis wasalso performed adjusting for HR/HER2 status. In an exploratory analysis,the marker with the greatest interaction (phosphorylated EGFR (Y1173))was selected to dichotomize patients optimally based on the data andassessed it in the context of the graduating signature by adding theEGFR Y1173-High patients to the HR−/HER2+ subtype and evaluating thetreatment effect in this ‘biomarker-positive’ group. Statisticalcalculations are descriptive (e.g. p-values are measures of distancewith no inferential content).

Methods

ISPY-2 TRIAL pre-treatment biopsy specimens were subjected to LCM toprocure tumor epithelium for reverse phase protein microarray (RPPAanalysis). Approximately 10,000 cells were captured for each of 168samples in the neratinib treatment and concurrent control arms. (107 Ntreatment arm, 64 control samples).

RPPA data was collected for 18 qualifying biomarkerproteins/phosphoproteins related to HER family signaling: AKT S473, AKTT308, EGFR, EGFR Y1068, EGFR Y1148, EGFR Y1173, EGFR Y992, ERBB2, ERBB2Y1248, ERBB3, ERBB3 Y1289, ERK1/2 T202/Y204, Heregulin, mTOR, mTORS2448, PI3K p85 Y458/p55 Y199, PTEN S380, and SHC Y317.

Reverse-phase Protein Microarray Construction

A full description of the technology and associated methodology isdiscussed in Example 3. Briefly, lysates (approx 40 microliters) fromLCM procured cells or cell culture were loaded into 384-well plates inserial dilutions (neat, 1:2, 1:4, 1:8 and 1:16) with negative controlwells containing lysis buffer only. Each dilution series was printed intriplicate onto nitrocellulose-coated glass slides (Schleicher andSchuell Bioscience, Keene, N.H., USA) using a custom solid pin contactarrayer (Aushon 2470, Aushon Biosystems, Boston Mass.). In order tobridge between slides and for quantitation, controls and calibratorswere printed on every slide. These consisted of lysates from A431 celllines (+/−EGF stimulation, Hela+/−pervanadate, Jurkat+/−calyculin,Jurkat+/−Fas ligand; BD Pharmingen, San Diego, Calif.,). Allphosphorylation and protein endpoints from the RPPA were normalized tototal protein. This was performed by staining with Sypro Ruby ProteinBlot Stain (Molecular Probes, Eugene, Oreg., USA) on one representativeslide from each array run. This technique The slides were analyzed usinga NovaRay™ Imaging System (Alpha Innotech, San Leandro, Calif.).

Reverse Phase Protein Microarray Data Analysis

Values obtained by the measurement of laser capture microdissected tumorepithelium using the reverse phase protein microarray technologyquantitatively measured the activation/phosphorylation level as well asthe total protein level of hundreds of signaling proteins at once.Values were obtained by comparison of values obtained from each patienttumor sample compared to calibrators that are printed on the same arrayso that a patient value was calculated and determined by a mathematicaltransformation to a relative intensity value if using referencestandards (reference units or RU) or specific amounts of an analyte wereused to make up the calibration curve to generate specific amounts orconcentrations (e.g. micrograms or micrograms/milliliter).

Laser Capture Microdissection (LCM)

LCM was performed in order to obtain pure populations of input materialfor cell signaling analysis. Using LCM (Pixcell II, Arcturus Bioscience,Mountain View, Calif.), approximately 20,000 cells (approx 5000 lasershots) were obtained from tumor epithelium derived from separatealternate and concurrent cryostat sections. Tissue processing andpreparation of tissue lysates have been described in the literature.

Immunostaining.

RPPA were immunostained on an automated slide stainer (Dako,Carpinteria, Calif.) using a biotinyl-linked catalyzed signalamplification system (CSA, DAKO) and a near-infrared dye coupledsecondary antibody for florescent detection (LICOR Biosystems, LincolnNebr.). All antibodies were pre-validated by Western blot and antibodyconcentrations were optimized using test arrays similar to thoseincluded in the study.

Image Analysis

Stained slides were scanned using a Novarray fluorescent laser scannerwith cooled CCD (Alpha Innotech). The florescent images were analyzedusing MicroVigene software (VigeneTech, Boston, Mass.). Briefly, asingle intensity value was determined for each endpoint and each patientby mathematically determining linear dynamic range of the sample, andextrapolation of the intensity value to the calibrator. The antibodyintensity was normalized to total protein and to a secondary antibodyalone control, and the replicates for each case were averaged. Theintensity values for each antibody and case are imported into MicrosoftExcel (Microsoft, Redmond, Wash., USA).

Statistical Analyses

Logistic regression was used to assess association with pCR in thecontrol and neratinib treated populations individually. Relativebiomarker performance between arms (biomarker×treatment interaction) wasassessed using a logistic model(pCR˜treatment+biomarker+treatment×biomarker). Analysis was alsoperformed adjusting for HR/HER2 status(pCR˜treatment+biomarker+treatment:biomarker+HR status+HER2 status).Permutation testing was used to determine statistical significance.

Bayesian Analyses

Bayesian analysis was completed using the MCMC simulation based on I-SPY2 data with the following model:pCR˜HR+HER2+biomarker+treatment+treatment*HR+treatment*HER2+treatment*biomarker.No multiple comparison adjustments were applied. The Bayesian analyticalengine developed by Don Berry (Yee et al J Clin Oncol. 2012 Dec. 20;30(36):4584-6), is a mathematical approach that evaluates the clinicalperformance of a drug (in this case, neratinib) in both the treatmentarm (in this case tumor shrinkage estimates by size changes observed byMRI), coupled with complete pathological response (pCR) as measured asthe final response determinant, and the response of the predicatetherapy in the control arm. This evaluation determined success of agiven therapy for a Phase III trial, and in this case, it was determinedthat phosphorylation of EGFR at Y1173 was not only statisticallysignificant at predicting or assessing response to neratinib in allpatients regardless of hormone receptor levels and HER2 levels (HER2+,HER2− and HR+ and HR−), but when patients with high levels ofphosphorylated EGFR (Y1173) were added to the HR−/HER2+ subgroup, theprevalence of ‘biomarker-positive’ patients increased by 50%, whileincreasing the predicted probability of Phase III success to 90%.Moreover, when EGFR Y1173 was used as a single marker, a statisticallysignificant increase in Phase III success was determined for allpatients compared to HER2+/HR− status as measured by immunohistochemicalmethods. (93% vs 87%).

Results

A number of HER family biomarkers associated with response in theneratinib treatment arm (see Table 1):

TABLE 1 Association With pCR in Neratinib Treated and Concurrent ControlPopulations P-value Endpoint Control Neratinib AKT S473 0.986 0.475 AKTT308 0.591 0.580 EGFR total 0.098 0.185 EGFR Y1068 0.252 0.015 EGFRY1148 0.360 0.784 EGFR Y1173 0.291 0.018 EGFR Y992 0.291 0.001 ERBB2total 0.847 0.019 ERBB2 Y1248 0.247 0.007 ERBB3 total 0.138 0.498 ERBB3Y1289 0.968 0.034 ERK1/2 T202/Y204 0.895 0.557 Heregulin total 0.8600.611 mTOR S2448 0.786 0.328 mTOR total 0.229 0.135 PI3K p85 Y458/p55Y199 0.943 0.238 PTEN S380 0.733 0.976 SHC Y317 0.269 0.025Seven (7) HER pathway markers (EGFR Y1068, EGFR Y1173, EGFR Y992, ERBB2total, ERBB2 Y1248, ERBB3 Y1289, SHC Y317) were associated with responsein the Neratinib (all patients without regard to HER2/HR status) but notthe control arm (see FIG. 1). However, the difference in performancebetween arms did not reach significance by permutation testing.Adjusting for HR/HER2 status, EGFR Y1173 shows a significantbiomarker×treatment interaction (p=0.049). In an exploratory analysis,patients were dichotomized by their EGFR Y1173 levels and evaluated thedistribution of pCR rates (Table 2).

TABLE 2 Distribution of pCR rates within EGFR Y1173 Low/High groupsstratified by the graduating HR−/HER2+ signature in each treatment arm.Neratinib (N = 106) Control (N = 62) EGFR EGFR EGFR EGFR Y1173 Y1173Y1173 Y1173 Low High Low High (N = 31) (N = 75) (N = 29) (N = 33)HR−/HER2+ 0/4 (0%) 12/18 (67%)  1/1 (100%)  1/5 (20%) (N = 28) NotHR−/HER2+ 3/27 (11%) 24/57 (42%) 5/28 (18%) 5/28 (18%) (N = 140)

The odds ratio (OR) between EGFR Y1173 groups in the N relative tocontrol arm was 10.1. When EGFR Y1173 High patients were added to thegraduating HR−/HER2+ subset, the OR associated with treatment is 3.2 andis comparable to that in the HR−/HER2+ signature (OR: 2.1), whileincreasing the prevalence of biomarker-positive patients by ˜50%. FIG. 2provides Bayesian evaluation of EGFR Y1173 as a biomarker of neratinibresponse and Table 4 below provides estimates of probability ofNeratinib superiority to control and probability of Phase III success.

TABLE 3 Bayesian Estimates of Probability of Neratinib Superiority toControl and Probability of Phase III Success Prob Ph III Prob > CTRLSuccess HR−/HER2+ 0.97 0.87 EGFR Y1173 High 0.99 0.93 HR−/HER2+ OR EGFRY1173 High 0.99 0.9 HR−/HER2+ AND EGFR Y1173 High 0.99 0.95Measurements of the phosphorylation levels of EGFR (Y1173, Y992, Y1068)along with total and activated HER2 (Y1248) and SHC (Y317) werepositively associated with response to neratinib combination therapy butnot standard of care treatment. Levels of EGFR Y1173, but not totalEGFR, are associated with response to neratinib combination therapy butnot standard of care. As a single biomarker, dichotomized in anexploratory analysis using data from this trial, EGFR Y1173 appears tohave a 93% probability of Phase III success, which exceeds that of theHR−/HER2+ graduating signature. When patients with high levels ofphosphorylated EGFR (Y1173) are added to the HR−/HER2+ subgroup, theprevalence of ‘biomarker-positive’ patients increased by 50%, whileincreasing the predicted probability of Phase III success to 90%. Theseexploratory analyses suggest that EGFR Y1173 phosphoprotein representeda possible biomarker for expanding the predicted responding patientpopulation.

Analysis shown here found that 7 HER pathway markers (EGFR Y1068, EGFRY1173, EGFR Y992, ERBB2 total, ERBB2 Y1248, ERBB3 Y1289, SHC Y317) wereassociated with response to neratinib but not the control arm across allpatients treated regardless of their HER2 and HR status. Moreover,statistical analysis further revealed that EGFR Y1173 can be used torefine the specific HER2+HR− cohort where neratinib showed clinicalsuccess. The ability of these 7 markers to add to and refine theHER2+HR− cohort could provide significant improvement of patientselection for neratinib treatment and be valuable companiondiagnostic/theranostic markers.

Example 2

In the same manner as described in Example 1, the following biomarkerswere identified as being predictive for a positive outcome for thesubjects of Example 1, pCR. The protein levels were measured for one ormore of the biomarkers disclosed herein, the measured protein levelswere compared to the protein levels from control samples (which formedthe baseline value for each biomarker) ALK.Y1586; ALK.Y1604;AMPKb1.S108; Caspase.7, cleaved D198; Cyclin.B1 total; Cyclin.D1.total;EGFR.Y1068, EGFR.Y1173, EGFR.Y992, eIF4G.S1108; IGFBP5.total;ERBB2.total; ERBB2.Y1248; ERBB2.Y877; ERBB3.Y1289; ERBB4.Y1284;FAK.Y576.Y577; JAK1.Y1022 and Y1023; JAK2.Y1007; MEK1.2.S217 and S211;MET.Y1234 and Y1235; p70S6K.T389; p70S6K.T412; PI3K.p85.Y458.p55. andY199; AKT S473; AKT T308; EGFR total; EGFR Y1148; ERBB3 total; ERK1/2T202/Y204; Heregulin total; mTOR S2448; mTOR total; PTEN S380; SHC Y317;PARP.total; PDGFRa.Y754; RET.Y905; RTK.ROR1.total; SHC.Y317; STAT5.Y694;VEGFR2.Y996; X4EBP1.S65; AMPKa1.S485; A.RAF.S299; IGF1R.Y1131. andIR.Y1146; MCSFR.Y732; A.RAF.S299; LC3B.total; TIE2.Y992; aJAK.STAT.pathway score (which is the sum of the measurements for JAK1Y1022/Y1023, JAK1 Y1007, STAT1 Y701 STAT2Y727, STAT3 Y705, STAT5 Y694),a mTOR pathway score (which is the sum of the measurements for 4EBP1S65; eIF4E S209; eIF4G S1108; eIF4G S1108; eIF4G S1108; mTOR S2448;p70S6K S371; p70S6K T389; p70S6K T412; S6RP S240/S244); a HER Familypathway score (which is the sum of the measurements for EGFR Y1068; EGFRY1173; EGFR Y992; ERBB2 Y1248; ERBB3 Y1289; FAK Y576/Y577; SHC Y317;STAT5 Y694; RET Y905); and a RTK pathway score (which is the sum of themeasurements for ALK Y1604; EGFR Y1068; EGFR Y1173; EGFR Y992; ERBB2Y1248; ERBB3 Y1289; FAK Y576/Y577; SHC Y317; STAT5 Y694; ERBB2 Y877;ERBB4 Y1284; MET Y1234-Y1235; ROS Y2274; RET Y905), or combinationsthereof. FIGS. 5-10, inclusive of alphabetically labeled portions, showthe analysis of these biomarkers as being predictive for a positiveresponse to administration of neratinib for treatment of breast cancer.

Example 3

Reverse Phase Protein Microarray (RPPA)

The Reverse Phase Protein Microarray (RPPA) technology was developed toaddress the analytical challenges of the sandwich and forward phaseprotein arrays (e.g. mismatch of sandwich antibody affinity, imprecisionwithin and between analytes, and poor sensitivity). The platform wasdesigned to enable non-subjective, quantitative, multiplexed analysis ofspecific forms of cellular proteins (e.g. phosphorylated,unphosphorylated, and cleaved) from a limited amount of starting sample,such as with a fine needle aspirate or laser capture microdissected(LCM) cellular material to procure pure populations of the target cellsof interest. Particularly suited for clinical tissue samples, RPPA usesa single antibody directed against the epitope of interest.

A key attribute of the RPPA was the ability to quantitatively measurehundreds of signaling proteins concomitantly from only a few thousandcells, thus providing a critical means of broad-scale cell signalinganalysis directly from tissue samples, cell culture models, and animaltissues from pre-clinical studies. The RPPA technology was optimized forroutine clinical sample analysis (1-10), and is currently employedwithin the CAP/CLIA complaint proteomics laboratory within the Centerfor Applied Proteomics and Molecular Medicine at George MasonUniversity. No other technology can measure the activity of as manysignaling proteins at once from such small amounts of input material.

The Reverse Phase Protein Microarrays (RPPA) immobilizes the test sampleanalytes (eg. lysate from laser capture microdissected cells) on thesolid phase. An analyte-specific ligand (e.g. antibody) is applied inthe solution phase (Capture). Bound antibodies are detected by secondarytagging and signal amplification (Signal Generation).

The RPPA method has the following major steps.

Overview of RPPA

A selection of peer-reviewed publications contains extensive detaileddescription of the basic core components RPPA methodology (Seereferences). The RPPA format immobilizes an individual test sample ineach array spot. See FIG. 3. An array can be comprised of up to hundredsof patient samples or cellular lysates. Each array was incubated with asingle primary antibody and a single analyte end point was measured.Since RPPAs maintained the concentration of the input sample, thesensitivity was greater as compared with a forward phase, (e.g. antibodyarray) probed with the same small number of input cells.

With the RPPA technology, serial dilutions were printed of each sample,control or standard, to maintain sample concentration. Each spotcontained a bait zone measuring only a few hundred microns in diameter.The detection probe can be tagged and signal amplified independentlyfrom the immobilized analyte protein. Coupling the detection antibodywith highly sensitive amplification systems yielded detectionsensitivities to fewer than 1,000 to 5,000 molecules per spot with goodlinearity (correlation coefficient or R²=0.990-0.999) andinter-experiment precision (R²=0.973). Between run and within runanalytical precision was between a 3-13% CV (coefficient of variation)(7).

The RPPA technology has been developed and optimized for performance asa fluorescent-based calibrated assay, generally identical in design andanalysis to standard ELISA or standard clinical immunoassays. As acalibrated assay, each assay consists of:

-   -   1. Experimental patient samples printed in triplicate two-spot        dilutions (neat and 1:4)    -   2. High, medium, and low controls printed in triplicate two-spot        dilutions (e.g., neat and 1:4)    -   3. A calibrator, consisting of a 6-10-point curve whereby the        analyte of interest is decreasing in concentration in the        background of a constant protein concentration.

The analyte concentration was thereby determined by extrapolation to anon-parametrically determined curve fit of the calibration curve andreported in relative fluorescent units.

Sample Preparation for Microarray

In order to prepare the sample for arraying, proteins were extractedfrom the LCM polymer cap as a whole cell lysate using a heated sodiumdodecyl sulfate-based lysing solution which produced a denatured lysatesuspended in the sample/extraction buffer. The optimal extraction bufferfor extracting proteins from tissue cells that have been procured byLCM, with the purpose of performing reverse phase protein arrays,consisted of a detergent, denaturing agent and buffer. This buffer wasan efficient denaturing extraction buffer for the extraction andsolubilization of cellular proteins from fixed and frozen tissue. Anarray layout grid was used to determine exact placement of sample andcontrol cell lysates on the printed microarray. Cell lysate solutionsfor each sample, of a known volume and concentration were loaded into384 well microtiter plates. Microtiter plates were specifically labeledand loaded into the well plate hotel in the correct order.

The RPPA used slides coated with nitrocellulose. This type of slide waschosen for its high binding capacity, high surface area, minimum effecton protein structure, and intrinsically low background signal. For theprinting run, up to 100 slides, (10 slides/platen and 10 platens withinthe Substrate Hotel), were loaded into the Aushon 2470 Arrayer at atime.

Array Preparation

The Aushon 2470 Arrayer had a general software program to manage theprinting process. The program enabled customization of array printing,with parameters such as top and left offset of printing,depositions/feature, slide lot number, number of replicates, dwell timefor pins, total number of immersions, maximum number of extractions andwash sequences.

Calibration of Values

As shown in FIG. 3, each array contained a printed calibrator(s), aseries of cell lystates derived from cells treated with a variety ofmitogens such that broad pathway activation has been achieved. Thecalibrator(s) consisted of 6-10 dilutions of whole cell lysates fromstimulated and unstimulated cells (eg. HeLa cells treated and untreatedwith pervandate for 30 minutes; jurkat cells treated and untreated withcalyculin for 30 minutes; A431 cells treated and untreated with EGF for30 minutes) pre-mixed in various ratios such that the total protein inany spot does not change, but the phospho-analyte changes in apredictable and defined concentration. Another type of calibrator can beprepared by spiking-in known amounts of recombinant protein or peptidesthat correspond to the target analyte and react specifically withantibodies directed to the target protein into a lystate that does notcontain the target analyte. The exact same calibrator was printed onevery single slide. The defining characteristic of this calibrator wasthat protein concentration does not vary but staining intensity did.Much like a clinical assay run in a diagnostic laboratory, eachexperimental value was extrapolated to a non-parametric curve fit of thecalibrator within the region that spanned the dynamic range of thepopulation such that results can be compared over time and acrossarrays. The calibrator was defined either in absolute amounts (if theanalyte concentration is known), or in relative units (RUs) if theabsolute amount of the analyte within the calibrator is not known. Mostapplications will use RU calibration units.

Data Normalization

Each protein analyte value was normalized to the total amount of proteinprinted on that spot by first incubated the slide with a florescentstain (Sypro Ruby Blot Stain, Molecular Probes, Eugene Oreg.) that bindsto proteins without bias and did not interfere with subsequent antibodybinding. The protein loading value is also obtained by a calibratedassay technique. A protein calibration curve of the exact same samplewas printed on every slide. This total protein calibrator consisted of aprotein lysate, which upon dilution, spanned the linear dynamic range ofprotein concentration. Each sample value was then extrapolated to thecalibrator. Consequently, while the total amount of protein may vary inany given sample compared to each other—thus affecting phopsho-proteinmeasurements for each sample, this variance was greatly minimized bysuch a normalization procedure.

Blocking Procedure

Once arrays were printed and stained for total protein, slides underwenta blocking procedure. Casein based solutions provided a uniform proteinsolution capable of binding to non-antigenic sites on nylon, PVDF andnitrocellulose membranes. Casein blocked these sites, inhibiting bindingof antibody. This resulted in reduced background staining for reversephase protein arrays.

Staining and Image Acquisition

Arrays were probed using an antibody specific for the phospho-protein,or any protein analyte. Over 350 phosphoproteins have been extensivelypre-validated for specificity using Western blotting and peptidecompetition. A Dako Cytomation Autostainer (FDA approved for theHercepTest) was used to perform the staining procedure. This includedthe processes of incubation with primary antibody, specific for theanalyte of interest as well as incubation with secondary antibody. Asignal was generated using a near-IR fluorescent dye (LICOR Biosciences)that was coupled to the secondary antibody. The RPPA used a fluorometricimage capture processing system (e.g. NovaRay, Alpha Innotech) for imageacquisition. The system measured the sample's fluorescence intensityvalue, subtracted the background, normalized the result to the totalprotein, and extrapolated the value to the non-parametrically fitcalibration curve to generate a final intensity value. The median of thetriplicate values was reported.

Correlation of Calibrated Values with Clinical Outcomes

Calibrated values of patient samples were correlated with outcomesresults (discontinuous variables (alive v dead, long v short survival),or continuous variables (overall survival, disease free survival, timeto progression, etc). These values were usually reported in days, weeksor months. Statistical analysis was used for the correlative findings.Parametric (e.g., Student t-test) or non-parametric (e.g. Wilcoxon RankSum) of mean comparison was used, Kaplan Meir and ROC curves were usedto uncover relationships between continuous clinical variables andcontinuous calibrated values. Optimally, any optimal cutpoint found bysuch analysis should be tested in independent study sets using ROC andor KM type analysis.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the method and compositions described herein. Suchequivalents are intended to be encompassed by the following claims.

What is claimed is:
 1. A method for treating breast cancer regardless ofER, PR and/or HER2 status, comprising, treating a subject identified ashaving breast cancer regardless of ER, PR and/or HER2 status with atleast one tyrosine kinase inhibitor (“TKI”) that targets EGFR and HER2,wherein the efficacy of the at least one TKI has been determined by a)measuring protein levels of one or more biomarkers in cellular samplesfrom the subject prior to treatment with the TKI, and b) comparing themeasured protein levels of the one or more biomarkers from the subjectto a baseline value for the respective one or more biomarkers, whereinan elevated or decreased level of the proteins of the one or morebiomarkers indicates that the subject is a responder to the TKI, whereina biomarker is selected from one or more of the following: ALK.Y1586;Cyclin.B1 total; EGFR.Y1068; EGFR.Y1173; EGFR.Y992; eIF4G.S1108;ERBB2.total; ERBB2.Y1248; ERBB2.Y877; ERBB4.Y1284; RET Y905; SHC Y317;an mTOR pathway score (which is the sum of the measurements for 4EBP1S65; eIF4E S209; eIF4G S1108; eIF4G S1108; eIF4G S1108; mTOR S2448;p70S6K S371; p70S6K T389; p70S6K T412; S6RP S240/S244); a HER Familypathway score (which is the sum of the measurements for EGFR Y1068; EGFRY1173; EGFR Y992; ERBB2 Y1248; ERBB3 Y1289; FAK Y576/Y577; SHC Y317;STAT5 Y694; RET Y905); and a RTK pathway score (which is the sum of themeasurements for ALK Y1604; EGFR Y1068; EGFR Y1173; EGFR Y992; ERBB2Y1248; ERBB3 Y1289; FAK Y576/Y577; SHC Y317; STAT5 Y694; ERBB2 Y877;ERBB4 Y1284; MET Y1234-Y1235; ROS Y2274; RET Y905), or combinationsthereof.
 2. The method of claim 1, wherein measurements are made withreverse phase protein microarray, ELISA, immunohistochemistry, westernblot, dot blot, mass spectrometry, mass cytometry, antibody array, orsuspension bead array.
 3. The method of claim 1, wherein the subject isidentified as having Stage II/III breast cancer.
 4. The method of claim1, wherein the subject has been treated with chemotherapy.
 5. The methodof claim 1, wherein the one or more proteins is phosphorylated EGFRY1173, and wherein the cellular sample comprises breast cancer cells ofsubtype HER2+/HR−.
 6. The method of claim 1, wherein the TKI comprisesneratinib, affatinib, lapatinib, pan-HER inhibitors or pan-EGFRinhibitors.
 7. The method of claim 1, wherein the subject is a humanpatient.
 8. The method of claim 5, wherein the subject is a humanpatient.
 9. The method of claim 6, wherein the subject is a humanpatient.